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H.  GLENN  BELt 


A      TEXT  -  BOOK      OF 

BACTERIOLOGY 


.1H3 


(^jjJjm'M^ 


A    TEXT-BOOK    OF 

B ACTE  RI OLOGY 

A  PRACTICAL  TREATISE  FOR  STUDENTS^  (  /  j 
AND    PRACTITIONERS    OF   MEDICINt:  ,  C\ 

BY 

PHILIP  HANSON^SS,  Jr.,  M.D. 

LATE   PROFESSOR   OF   BACTERIOLOGY,    COLLEGE   OF    PHYSICIANS   AND   SURGEONS, 
COLUMBIA    UNIVERSITY,    NEW   YORK    CITY 

AND 

HANS   ZINSSER,   M.D. 

PROFESSOR   OF   BACTERIOLOGY,    COLLEGE    OF    PHYSICIANS    AND   SURGEONS,    COLUMBIA    UNI- 
VERSITY,   NEW    YORK   city;    BACTERIOLOGIST    TO    THE   PRESBYTERIAN    HOSPITAL; 
FORMERLY    PROFESSOR    OF    BACTERIOLOGY    AND    IMMUNITY,    STANFORD 
UNIVERSITY,    CALIFORNIA;    MAJOR,    MEDICAL   OFFICERS' 
RESERVE   CORPS,    U.  S.  A. 


WITH   A   SECTION   ON    THE   PATHOGENIC    PROTOZOA 

BY 

FREDERICK  F.   RUSSELL,   M.D. 

MAJOR   MEDICAL   CORPS,    U.  S.  A.;    FORMERLY    PROFESSOR   OF   BACTERIOLOGY   AND 
PATHOLOGY,    ARMY    MEDICAL   SCHOOL   AND   GEORGE   WASHINGTON    UNIVERSITY 


WITH  ONE  HUNDRED  AND  NINETY-EIGHT  ILLUSTRATIONS  IN  THE 
TEXT,  SOME  OF   WHICH  ARE  COLORED 


FOURTH  EDITION 


NEW    YORK   AND    LONDON 

D.    APPLETON    AND    COMPANY 

1919 


154103 


I 

V'  v 

4'i 

COPTRIGHT,   1910.    1914,   1916  AND   1918, 
D.   APPLETON  AND   COMPANY 


Printed  in  New  York,  U.  S.  A. 


PREFACE   TO   THE   FIRST   EDITION 

The  volume  here  presented  is  primarily  a  treatise  on  the  funda- 
mental laws  and  technique  of  Bacteriology,  as  illustrated  by  their 
application  to  the  study  of  pathogenic  bacteria. 

So  ubiquitous  are  the  bacteria  and  so  manifold  their  activities 
that  Bacteriology,  although  one  of  the  youngest  of  sciences,  has 
already  been  divided  into  special  fields— Medical,  Sanitary,  Agricul- 
tural, and  Industrial — having  little  in  common,  except  problems  of 
general  bacterial  physiology  and  certain  fundamental  technical  pro- 
cedures. 

From  no  other  point  of  approach,  however,  is  such  a  breadth  of 
conception  attainable,  as  through  the  study  of  bacteria  in  their  rela- 
tion to  disease  processes  in  man  and  animals.  '  Through  such  a 
study  one  must  become  familiar  not  only  with  the  growth  character- 
istics and  products  of  the  bacteria  apart  from  the  animal  body,  thus 
gaining  a  knowledge  of  methods  and  procedures  common  to  the  study 
of  pathogenic  and  non-pathogenic  organisms,  but  also  with  those 
complicated  reactions  taking  place  between  the  bacteria  and  their 
products  on  the  one  hand  and  the  cells  and  fluids  of  the  animal  body 
on  the  other — ^reactions  which  often  manifest  themselves  as  symptoms 
and  lesions  of  disease  or  by  visible  changes  in  the  test  tube. 

Through  a  study  and  comprehension  of  the  processes  underl5dng 
these  reactions,  our  knowledge  of  cell  physiology  has  been  broadened, 
and  facts  of  inestimable  value  have  been  discovered,  which  have 
thrown  light  upon  some  of  the  most  obscure  problems  of  infection 
and  immunity  and  have  led  to  hitherto  unsuspected  methods  of 
treatment  and  diagnosis.  Thus,  through  Medical  Bacteriology — that 
highly  specialized  offshoot  of  General  Biology  and  Pathology — have 
been  given  back  to  the  parent  sciences  and  to  Medicine  in  general 
methods  and  knowledge  of  the  widest  application. 

It  has  been  our  endeavor,  therefore,  to  present  this  phase  of  our 
subject  in  as  broad  and  critical  a  manner  as  possible  in  the  sections 


vi  PREFACE 

dealing  with  infection  and  immunity  and  with  methods  of  biological 
diagnosis  and  treatment  of  disease,  so  that  the  student  and  practi- 
tioner of  medicine,  by  becoming  familiar  with  underlying  laws  and 
principles,  may  not  only  be  in  a  position  to  realize  the  meaning  and 
scope  of  some  of  these  newer  discoveries  and  methods,  but  may  be 
in  better  position  to  decide  for  themselves  their  proper  application 
and  limitations. 

We  have  not  hesitated,  whenever  necessary  for  a  proper  under- 
standing of  processes  of  bacterial  nutrition  or  physiology,  or  for 
breadth  of  view  in  considering  problems  of  the  relation  of  bacteria 
to  our  food  supply  and  environment,  to  make  free  use  of  illustrations 
from  "the  more  special  fields  of  agricultural  and  sanitary  bacteriology, 
and  si)me  special  methods  of  the  bacteriology  of  sanitation  are  given 
in  the  last  division  of  the  book,  dealing  with  the  bacteria  in  relation 
to  our  food  and  environment. 

Iri  conclusion  it  may  be  said  that  the  scope  and  arrangement  of 
subjects  treated  of  in  this  book  are  the  direct  outcome  of  many  years 
of  experience  in  the  instruction  of  students  in  medical  and  in  advanced 
university  courses  iii  bacteriology,  and  that  it  is  our  hope  that  this 
volume  may  not  only  meet  the  needs  of  such  students  but  may  prove 
of  value  to  the  practitioner  of  medicine  for  whom  it  has  also  been 
written. 

It  is  a  pleasure  to  acknowledge  the  courtesy  of  those  who  furnished 
us  with  illustrations  for  use  in  the  text,  and  our  indebtedness  to  Dr. 
Gardner  Hopkins  and  Professor  Francis  Carter  Wood  for  a  number 
of  the  photomicrographs  taken  especially  for  this  work. 

P.H.  H.,  Jr., 
H.  Z. 


PREFACE  TO   THE   SECOND   EDITION 

Inquiry  in  the  field  of  bacteriology  is  so  active  at  the  present  day 
that  no  general  text-book  can  maintain  its  usefulness  long  without 
frequent  revision.  In  preparing  the  second  edition  of  this  book  it  has 
been  our  purpose  to  correct  omissions  and  to  incorporate  the  more  im- 
portant researches  of  the  last  three  years,  rather  than  to  alter  exten- 
sively the  plan  of  the  text.  From  the  wealth  of  material  which  these 
years  have  brought,  we  have  attempted  to  glean  those  facts  which 
have  seemed  to  us  most  important  and  most  directly  bearing  upon 
medical  problems,  since  this  book  was  planned,  from  the  beginning, , to 
meet  especially  the  needs  of  the  student  of  infectious  disease. 

The  most  extensive  changes  and  additions  have  been  made  in  the 
chapters  on  streptococci,  tuberculosis,  plague,  leprosy,  syphiHs,  rabies, 
and  pohomyelitis.  Short  sections  on  typhus  fever,  on  the  plague- 
hke  disease  of  rodents,  and  on  rat  leprosy  have  been  added,  and  we  have 
inserted  a  tabulation  of  our  knowledge  of  filtrable  virus,  adapted 
largely  from  the  summary  recently  published  by  Wolbach.  The  Ander- 
son and  McClintic  method  for  the  standardization  of  disinfectants, 
and  Churchman's  recent  work  on  anihn  dyes  and  bacterial  growth, 
have  been  added.  Many  minor  corrections  and  additions  have  been 
made  throughout  the  text.  In  preparing  these  changes,  valuable  as- 
sistance has  been  given  us  by  Dr.  J.  Gardner  Hopkins,  Associate  in 
Bacteriology  at  Columbia  University,  and  many  helpful  suggestions 
have  been  made  by  Drs.  Dwyer  and  Bliss. 

It  has  been  gratifying  to  note  how'  much  of  the  work  which  seemed 
to  us  particularly  valuable  and  enhghtening  has  emanated,  during  these 
three  years,  from  American  laboratories. 

We  have  purposely  omitted  making  any  extensive  changes  in  the 
section  on  immunity.  The  function  of  this  part  of  the  book  is  to  give 
the  beginner  a  basis  for  further  reading  and  introduce  him,  as  simply  as 
possible,  to  the  difficult  problems  of  the  field.  We  have  felt  that  the 
addition  of  much  more  detail  and  theory  would  render  this  section  un- 
^uited  to  the  needs  of  a  general  text-book. 

It  is  a  sorrowful  necessity  that  this  revision  must  be  put  fprth  with- 

vii 


VUl 


PREFACE  TO  THE  SECOND  EDITION 


out  the  wise  counsel  of  one  of  its  authors.  Since  the  first  edition  of  this 
book  was  pubUshed  Prof.  PhiUp  Hanson  Hiss,  Jr.,  has  died.  By  his 
death  we  have  lost  a  dear  friend  and  a  valued  teacher,  and  American 
bacteriology  has  been  deprived  of  a  worker  who  was  entering  into  the 
most  brilliant  period  of  his  scientific  maturity. 

H.  Z. 
New  York,  1914 


PREFACE  TO  THIRD  EDITION 

The  need  for  a  new  edition  of  the  Text  Book  has  offered  a  welcome 
opportunity  for  the  addition  of  the  many  new  facts  revealed  by  investi- . 
gation  during  the  last  two  years.  A  thorough  revision  of  the  entire 
book  has  incidentally  been  made  since  experience  with  it  in  teaching, 
the  questions  of  students,  and  the  comments  of  associates  have  in- 
dicated sections,  here  and  there,  in  which  slight  changes,  elabora- 
tions or  omissions,  would  add  to  clearness  and  simplicity.  In  this,-  as 
in  the  choice  of  new  material,  the  writer  has  again  been  guided  chiefly 
by  the  desire  to  enhance  the  practical  usefulness  of  the  treatise  for  medi- 
cal students  and  workers  interested  primarily  in  infectious  diseases. 

In  the  section  on  Biology  and  Technique,  the  changes  have  been 
relatively  few.  To  the  chapter  on  culture  media,  there  have  been  added 
Kendall's  modification  of  Endo's  medium,  Petroff's  media  for  tubercle 
bacillus  cultivation,  Krumwiede's  brilliant  green  medium,  and  Russell's 
double  sugar  medium.  Many  other  minor,  yet,  in  our  opinion  impor- 
tant, alterations  have  been  made  in  this  chapter,  but  throughout  the 
book  only  such  methods  have  been  added  as  have  been  found  to  be 
actually  useful  in  our  own  practice  or  in  that  of  our  associates. 

The  section  on  Immunity  has  been  very  thoroughly  revised  in  order 
to  keep  within  the  same  small  space  a  more  accurately  modern  pres- 
entation of  this  complicated  subject.  It  has  not,  of  course,  been  possible 
to  cover  this  field  as  thoroughly  as  ijb  might  be  covered  in  a  volume 
separately  devoted  to  the  subject,  but  the  outline  here  given  is  intend- 
ed mainly  to  furnish  a  sound  foundation  for  further  study. 

In  the  chapter  on  the  pathogenic  microorganisms  themselves,  the 
most  extensive  changes  have  been  necessary  in  the  pneumococcus  sec- 
tion and  in  the  one  on  treponema  pallidum.  However,  all  the  chapters 
have  been  revised  with  care  and  many  small  additions  and  omissions 
made  where  new  facts  and  the  revision  of  older  opinions  have  made  this 
desirable. 

H.  Z. 
College  of  Physicians  and  Surgeons,  Columbia  University.  N.  Y. 

ix 


PREFACE  TO  THE  FOURTH  EDITION 

The  most  important  change  incorporated  in  the  fourth  edition  of 
our  Text-Book  is  the  section  on  Pathogenic  Protozoa  written  by- 
Major  Frederick  F.  Russell,  of  the  Medical  Corps  of  the  United 
States  Army.  When  the  first  edition  of  this  book  was  written  the  ad- 
dition of  a  section  on  Protozoa  was  seriously  considered,  but  was 
finally  omitted  because  it  was  the  desire  of  both  the  writers  at  that 
time  that  nothing  should  go  into  the  book  that  could  not  be  based 
on  personal  knowledge,  and,  since  neither  the  late  Dr.  Hiss  nor  the 
undersigned  had  worked  systematically  in  the  field  of  protozoology, 
it  was  thought  better  to  limit  the  book  to  our  own  field  of  bacteriology. 
The  book  has  since  then  gone  through  three  editions  and  it  has  be- 
come more  and  more  apparent  that  it  has  found  its  greatest  useful- 
ness as  a  text-book  for  medical  students  and  a  reference  book  for 
physicians  and  laboratory  workers.  For  these  purposes,  however, 
it  has  had  the  serious  defect  of  giving  no  information  about  such 
microorganisms  as  the  malaria  plasmodia,  the  trypanosomes  and 
other  pathogenic  microorganisms,  which,  though  technically  protozoa, 
are  necessary  objects  of  interest  to  all  medical  laboratory  workers. 
It  has  appeared  to  us,  therefore,  that  the  practical  value  of  the  book 
would  be  greatly  enhanced  by  the  addition  of  a  short  treatise  on  the 
protozoa,  if  written  by  someone  who  had  been  in  touch  with  this  field, 
rather  from  the  practical  medical  point  of  view  than  from  that  of  the 
zoologist.  Major  Russell  has  kindly  consented  to  supply  this  de- 
ficiency in  a  section  which  makes  no  claim  to  zoological  complete- 
ness, but  is  written  with  the  definite  purpose  of  giving  medical 
workers  concise  information  concerning  the  important  pathogenic 


xii  PREFACE  TO  THE  FOURTH  EDITION 

species,  with  especial  consideration  of  their  common  occurrence,  the 
methods  of  their  detection  and  recognition,  and  correlation  to  the 
diseases  which  they  incite. 

In  other  respects  the  book  has  been  thoroughly  gone  over.  In  the 
section  on  Biology  and  T^ichnique  a  few  methods,  which  we  ourselves 
have  found  it  unnecessary  to  use,  have  been  omitted.  A  few  other 
methods  have  been  added  and  a  few  of  the  most  useful  ones  ampli- 
fied. Minor  changes /have  been  made  in  the  section  on  Immunity. 
^  The  chapter  on  Streptococcus  has  been  revised  and  considerable  addi- 
tions and  changes  have  been  made  in  the  chapters  on  the  Paratyphoid 
and  Typhoid '9^cilli.  The  more  recent  work  on  the  Schick  Test  and 
on  the  Det^mination  of  Virulence  of  the  Diphtheria  Bacillus  has 
been  incorporated.  Minor  changes  have  been  made  in  the  sections 
on  Bacteria  in  Water  and  Milk. 

Altogether  it  has  been  attempted  to  bring  the  material  up  to  date, 
especially  in  those  features  in  which  we  think  its  greatest  usefulness 
lies,|namely,  as  a  guide  for  the  actual  performance  of  bacteriological 
work.  It  is  a  pleasure  again  to  acknowledge  the  valuable  assistance 
of  Prof .  J.  G.  Hopkins  in  correcting  and  rearranging  the  book.   , 

Hans  Zinsser. 

:  College  of 'Physicians  and  Surgeons, 
Columbia  University, 
N^ew  York  City. 


CONTENTS 


SECTION    I 

THE    GENERAL    BIOLOGY    OF    BACTERIA    AND    THE 
TECHNIQUE   OF   BACTERIOLOGICAL    STUDY 

CHAPTER  PAGE 

I.  The  Development  and  Scope  of  Bacteriology  .."....       1 
IT     General    Morphology,     Reproduction,    and    Chemical    and 

Physical   Properties  of  the  Bacteria  ........      9 

III.  The    Relation    of   Bacteria    to    Environment,    and    Their 

Classification    , 25 

IV.  The  Biological  Activities  of  Bacteria 40 

V.  The  Destruction  of  Bacteria 62 

VI.  Methods  Used  in  the  Microscopic  Study  and  Staining  of 

Bacteria 93 

VII.  The  Preparation  of  Culture  Media 113 

VIII.  Methods  Used  in  the  Cultivation  of  Bacteria 141 

IX    Methods  Determining  Biological  Activities  of  Bacteria   ,  164 
X.  The     Bacteriological     Examination     of     Material     from 

Patients    ..-.•... 174 


SECTION   n 

INFECTION    AND    IMMUNITY 

CHAPTER  PAGE 

XI,  Fundamental  Factors  of  Pathogenicity  and  Infection  .     .  181 
XII.  Defensive  Factors  of  the  Animal  Organism 189 

XIII.  Toxins  and  Antitoxins 203 

XIV.  Production  and  Testing  of  Antitoxins 216 

XV.  Lysins,  Agglutinins,   Precipitins,   and  Other  Antibodies    ,  224 

XVI,  The  Technique  of  Serum  Reactions    .     .    .     .     .     ,     .     .     .  250 

xiii 


XIV  Contents 

CHAPTER  PAGE 

XVII.  Phagocytosis 275 

XVIII.  Opsonins,  Leucocyte  Extract,  and  Aggressins 281 

XIX.  Anaphylaxis  or  Hypersusceptibility    ........  295 

XX.  Facts  and  Problems  of  Immunity   in  their  Bearing  upon 

THE  Treatment  of  Infectious  Diseases 305 


SECTION    III 


PATHOGENIC   MICROORGANISMS 

CHAPTER  PAGE 

XXI.  The  Staphylococci  (Micrococci) 321 

XXII.  The  Streptococci 335 

XXIII.  DiPLOCOCCUS  PNEUMONIA 352 

XXIV.  MiCROCOcctrs    intracellularis    meningitidis  (Meningococcus)    .  371 
XXV.  DiPLOcoccus      GONORRHCE^      (Gonococcus) ,      Micrococcus      ca- 

tarRhalis,  AND  Other  Gram-negative  Cocci 380 

XXVI.  Bacilli   of  the   Colon-Typhoid-Dysentery  Group — Bacillus 

COLI   COMMUNIS, 388 

XXVII.  !0AciLLi   OF   THE  Colon-Typhoid-Dysentery  Group  (continued) 

—Bacillus  of  Typhoid  Fever 399 

XX  VIII.  Bacilli  of  the  Colon-Typhoid-Dysentery  Group  (continued) 
— Bacilli  Intermediate  between  the  Typhoid  and  Colon 

X)rganisms 428 

XXIX.  Bacilli   of  the   Colon-Typhoid-Dysentery  Group  (continued) 

— The  Dysentery  Bacilli 435 

XXX.  Bacillus  mucosus  capsulatus 447 

XXXI.  Bacillus  tetani '  .  456 

XXXII.  Bacillus  of  Symptomatic  Anthrax,  Bacillus  of  Malignant 
Edema,  Bacillus  aerogenes  capsulatus.  Bacillus  botu- 
LiNUS 465 

XXXIII.  The  Tubercle  Bacillus 479 

XXXIV.  The  Smegma  Bacillus  and  the  Bacillus  of  Leprosy  .     .     .  503 
XXXV.  Bacillus    diphtheria.    Bacillus    Hoffmanni,    and    Bacillus 

xerosis 512 

XXXVI.  Bacillus  mallei 528 

XXXVII.  Bacillus  influenzae  and  Closely  Related  Bacteria    .     .     .  536 


i 


CONTENTS  XV 

CHAPTER  PAOB 

XXXVIII. .  Bordet-Gengou    Bacillus,    Morax-Axenfeld    Bacillus,  Zur 

Nedden  Bacillus,  Ducrey  Bacillus 543 

XXXIX.  The    Bacilli    of   the   Hemorrhagic   Septicemia   Group  aj^d 

Bacillus  pestis 551 

XL.  Bacillus  anthracis  and  Anthrax ,     .  553 

XLI.  Bacillus    pyocyaneus 577 

XLII.  Asiatic  Cholera  and  the  Cholera  Organism 582 

XLIII.  Diseases  Caused  by  Spirochetes 592 

XLIV.  The  Higher  Bacteria 618 

XLV.  The    Yeasts 629 

XLVI.  Hyphomycbtes 635 


SECTION    IV 

EXANTHEMATA  AND  DISEASES  CAUSED  BY 
FILTRABLE  VIRUS 

CHAPTER  PAOK 

XLVII.  Rabies 646 

XLVIII.  Smallpox 657 

XLIX.  Acute  Anterior  Poliomyelitis 664 

L.  Yellow    Fever '.     .  668 

LI.  Measles,  Scarlet  Fever,  and  Foot-and-Mouth  Disease    .     .  675 


SECTION   V 

BACTERIA   IN   AIR,   SOIL,'   WATER,   AND   MILK 

CHAPTER  PAGE 

LII.  Bacteria  in  the  Air  and  Soil 683 

LIII.  Bacteria   in   Water 689 

tlV.  Bacteria   in   Milk   and   MiiiK   Products,    Bactewa   in   the 

Inpu^TIU?^     .    ..    •.    ..    ..    •.    .    n    ••.••*    ^    .    "I    ..    .  699 


xvi  CONTENTS 

SECTION  VI 
PATHOGENIC  PROTOZOA 

Maj.  Frederick  F.  Russell,  Med.  Corps,  U.  S.  Army 
chapteb  page 

INTRODUCTION 721* 

LV.  Class  I — Sarcodina  (Rhizopoda) 723 

LVI.  Class  II — Mastigophora  (Diesing) , 738 

.     LVII.  Class  III— Sporozoa      . 760 

-.JiVIII,  Class  IV— Infusoria .792 

tiiX.  Technique  of  Blood  Examinations  for  Protozoa     .     .     .    .    .  795 

\lNDEX  OF  AUTHORSn 799 

[DEX  OF  SUBJECTsX 811 


LIST  OF  ILLUSTRATIONS 

FIGURE  PAGE 

1.  Types  of  bacterial  morphology 10 

2.  Bacterial  capsules 12 

3.  Arrangement  of  bacterial  flagella 15 

4.  Various  positions  of  spores  in  bacterial  cell 17 

5.  Germination  of  spores * 17 

6.  Degeneration  forms  of  Bacillus  diphtheriae 19 

7.  Degeneration  forms  of  Bacillus  pestis       »     .  20 

8.  Hot-air  sterilizer 69 

9.  Arnold  sterilizer 70 

10.  Low-temperature  sterilizer 71 

11.  Autoclave 72 

12.  Lentz  formalin  apparatus       . 90 

13.  Breslau  formaldehyde  generator  and  section  of  same 91 

14.  Hanging  drop  preparation 94 

15.  Florence  flask 114 

16.  Erlenmeyer  flask 114 

17.  Petri  dish 115 

18.  Test  tubes,  showing  method  of  stoppering 116 

19.  Burette  for  titrating  media 117 

20.  Tubing  media 118 

21.  Media  in  tubes 119 

22.  Berkefeld  filter 120 

23.  Berkefeld  filter 121 

24.  Reichel  filter 122 

25.  Kitasato  filter       .......' 123 

26.  Maassen  filter,  for  small  quantities  of  fluid 124 

27.  Platinum  wires 142 

28.  Taking  plugs  from  tubes  before  inoculation 143 

29.  Inoculating 144 

30.  Pouring  inoculating  medium  into  Petri  dish 145 

31.  Streak  plate 147 

32.  Deep  stab  cultivation  of  anaerobic  bacteria 148 

33.  Deep  stab  cultivation  of  anaerobic  bacteria 149 

34.  Cultivation  of  anaerobes  in  fluid  under  albolin 150 

35.  Wright's  method  of  anaerobic  cultivation  in  fluid  media 151 

36.  Novy  jar 152 

37.  Wright's  method  of  anaerobic  cultivation  by  the  use  of  pyrogallic-acid 

solution 153 

38.  Jar  for  anaerobic  cultivation 154 

2  xvii 


xviii  LIST  OF  ILLUSTRATIONS 

FIGURE  PAGE 

39.  Apparatus  for  combining  the  methods  of  exhaustion,  hydrogen  replace- 

ment, and  oxygen  absorption 155 

40.  Simple  apparatus  for  plate  cultivation  of  anaerobic  bacteria 156 

41.  Incubator 157 

42.  Thermo-regulator 158 

43.  Thermo-regulator 158 

44.  Moitessier  gas-pressure  regulator 160 

45.  Variations  in  the  conformation  of  the  borders  of  bacterial  colonies    .     .  161 

46.  Wolffhiigel  counting-plate 162 

47.  Types  of  fermentation  tubes 165 

48.  Types  of  gelatin  liquefaction  by  bacteria 169 

49.  Intraperitoneal  inoculation  of  rabbit 170 

50.  Intravenous  inoculation  of  rabbit 170 

51.  Intraperitoneal  inoculation  of  guinea*pig 171 

52.  Guinea-pig  cage \ 171 

53.  Rabbit  cage J .  172 

54.  Blood-culture  plate  showing  streptococcus  colonies 179 

55.  Toxin  and  body  cell 206 

56.  Toxin  and  antitoxin       ....   / 214 

57.  Ehrlich's  conception  of^cel^receistors,  giving  rise  to  lytic  immune  bodies    227 

58.  Complement,  amboceptor  or  immune  body,  and  antigen  or  immunizing 

substance 227 

59.  Microscopic  agglutination  reaction 229 

60.  Macroscopic  agglutination 230 

61.  Ehrlich's  conception  of  the  structure  of  agglutinins  and  precipitins      .     .  238 
82.  The  structure  of  cell-receptors  and  immune  bodies,  according  to  Ehrlich's 

conception 239 

63.  Neisser  and  Wechsberg's  conception  of  complement  deviation     ....  246 

64.  Schematic  representation  of  complement  fixation  in  the  Bordet-Gengou 

reaction 248 

65.  Capillary  pipette  for  removal  of  exudate  in  doing  the  Pfeiffer  test    .     .256 

66.  Wright's  capsule  for  collecting  blood 284 

67.  Pipette  for  opsonic  work 285 

68.  Pipette  with  three  substances, — corpuscles,  bacteria,  and  serum,  as  first 

taken  up 285 

69.  Staphylococcus  pyogenes  aureus 322 

70.  Staphylococcus  colonies 323 

71.  Micrococcus  tetragenus 334 

72.  Streptococcus  pyogenes 336 

73.  Streptococcus  colonies  on  serum  agar 339 

74.  Streptococcus  colonies  from  blood  culture  on  blood-agar  plate     ....  345 

75.  Pneumococci 354 

76.  Pneumococci 354 

77.  Meningococcus,  pure  culture 372 

78.  Meningococcus  in  spinal  fluid 373 

79.  Meningococcus  culture  (streak  plate) .375 

80.  Gonococcus  pus  from  urethra 38J 


LIST  OF  ILLUSTRATIONS  xix 

MGTniB  PAGE 

8L  Gonococcus      .     .     .     .     .     .     ...     .     .     ...    .    .    .    ,     .    .     .  382 

82.  Gonococcus  colony 383 

83.  Bacillus  coli  communis 390 

84.  Bacillus  coli  communis  on  various  media 396 

85.  Bacillus  coli  communior  on  various  media 397 

86.  Bacillus  typhosus 400 

87.  Bacillus  typhosus,  showing  flagella 401 

88.  Surface  colony  of  Bacillus  typhosus  on  gelatin 402 

89.  Bacillus  coU;  deep  colonies  on  Hiss  plate  medium 407 

90.  Bacillus  typhosus;  deep  colonies  in  Hiss  plate  medium 408 

9L  Bacillus  typhosus;  colony  in  Hiss  plate  medium,  highly  magnified  .     .     .  409 

92.  Colon  and  typhoid  colonies  in  Hiss  plate  medium 410 

93.  Scheme  of  fermentations  of  the  dysentery-typhoid-colon-bacilli  in  carbo- 

hydrate serum-water  media 445 

94.  Bacillus  mucosus  capsulatus 448 

95.  Bacilli  of  rhinoscleroma 452 

96.  Bacillus  tetani 457 

97.  Young  tetanus  culture  in  glucose  agar 458 

98.  Older  tetanus  culture  in  glucose  agar 459 

99.  Bacillus  of  symptomatic  anthrax , 466 

100.  Bacillus  of  symptomatic  anthrax,  culture  in  glucose  agar 467 

101.  Bacillus  of  malignant  edema       469 

102.  Bacillus  of  malignant  edema,  culture  in  glucose  agar 470 

103.  Tubercle  bacilli  in  sputum 480 

104.  Culture  of  Bacillus  tuberculosis  in  flask  of  glycerin  bouillon  .....  486 

105.  Bacillus  diphtheriae 513 

106.  Colonies  of  Bacillus  diphtheriae  on  glycerin  agar 518 

107.  Bacillus  Hoffmanni 524 

108.  Colonies  of  Bacillus  Hoffmanni  on  agar 625 

109.  Bacillus  xerosis 626 

110.  Glanders  bacillus 529 

111.  Glanders  bacilU  in  tissue , 531 

112.  Bacillus  influenzae;   smear  from  pure  culture  on  blood  agar 537 

113.  Bacillus  influenzae;  smear  from  sputum 538 

114.  Colonies  of  influenza  bacillus  on  blood  agar 539 

V15.  Koch- Weeks  bacillus 542 

116.  Bordet-Gengou  bacillus 544 

117.  Morax-Axenfeld  diplo-bacillus 546 

118.  Bacillus  pestis • 655 

119.  Bacillus  pestis,  involution  forms      .     .     .     .     , 656 

120.  Bacillus  anthracis;  pure  culture  on  agar 564 

121.  Bacillus  anthracis,  in  kidney  tissue 565 

122.  Bacillus  anthracis,  in  spleen  tissue 566 

123.  Anthrax  colony  on  gelatin 567 

124.  Anthrax  colony  on  agar 568 

125.  Bacillus  subtilis    . 576 

126.  Cholera  spirillum       .....••... 583 


XX  LIST  OF  ILLUSTRATIONS 

FIGURE  .PAGE 

127.  Cholera  spirillum;  stab  cultures  in  gelatin,  three  days  old 586 

128.  Cholera  spirillum;  stab  culture  in  gelatin,  six  days  old 586 

129.  Spirochaete  pallida;  smear  from  chancre 594 

130.  Spirochaete  pallida,  in  spleen  of  congenital  syphilis 600 

131.  Spirochaete  paUida,  in  hver  of  congenital  syphilis 601 

132.  Spirochaete  of  relapsing  fever 605 

133.  Spirochaete  of  relapsing  fever 606 

134.  Spirochaete  of  relapsing  fever 607 

135.  Spirochaete  of  Dutton,  African  tick  fever 609 

136.  Smear  from  the  throat  of  a  case  of  Vincent's  angina 611 

137.  Throat  smear,  Vincent's  angina 612 

138.  Spirochaete  gallinarum 616 

139.  Cladothrix,  showing  false  branching 620 

140.  Streptothrix,  showing  true  branching 621 

141.  Actinomyces  granule  crushed  beneath  a  cover-glass 624 

142.  Actinomyces  granule  crushed  beneath  a  cover-glass 625 

143.  Branching  filaments  of  actinomyces  / 626 

144.  Yeast  cells    .  \ / 630 

145.  Mucor  mucedo      .     .     .     .     .   y 636 

146.  Mucor  mucedo    V.     .     '^^' 637 

147.  Mucor  mucedo .638 

148.  Mucor  ramosus 639 

149.  Penicillium  glaucmn 640 

150.  Aspergillus  glaucus 641 

151.  Thrush 642 

152.  Achorion  Schoenleinii 643 

153.  Method  of  drying  spinal  cord  of  rabbit  for  purposes  of  attenuation  .     .     .  653 

154.  Stegomyia  fasciata 671 

155.  Bacillus  bulgaricus 717 

156.  Endameba  histoljrtica     .6 724 

157.  Endameba  histolytica 725 

158.  Endameba  histolytica 729 

159.  Endameba  histolytica 730 

160.  Endameba  histolytica o 730 

161.  Endameba  coli  .     .     .     .     = 732 

162.  Endameba  coli  ....     » 733 

163.  Endameba  coli 734 

164.  Endameba  coli  cyst    .......     c     ... 735 

165.  Endameba  coli 736 

166.  Trichomonas  intestinalis 738 

167.  Lamblia  intestinalis.     Cyst  formation 739 

168.  Tr3rpanosoma  rotatorium  in  blood  of  frog 742 

169.  Trypanosoma  Lewsi 743 

170.  Most  important  trypanosomes  parasitic  in  vertebrates 746 

171.  Dourine.     Showing  swelling  of  genitalia  and  plaques  on  the  skin  .     .     .     .  749 

172.  Trypanosoma  avium  in  blood  of  common  wild  birds 750 

173.  Trypanosoma  avium  in  culture  on  blood  agar 751 


LIST  OF  ILLUSTRATIONS  xxi 

FIGURE  PAOB 

174.  Trypanosoma  garabiense 752 

175.  Tsetse  fly  (Glossina  palpalis) 753 

176.  Schizotrypanum  cnizi  in  human  blood 755 

177.  Schizotrypanum  cruzi  developing  in  tissues  of  guinea-pig 756 

178.  Leishmania  Donovani 757 

179.  Leishmania  infantum 759 

180.  Hsemoproteus  columbse 762 

181.  Proteosoma  precox  in  blood  of  field  lark 763 

182.  Midgut  of  culex  mosquito,  covered  with  oocysts  of  Proteosoma  praecox      .  764 

183.  Plasmodium  vivax 766 

184.  Plasmodium  vivax 766 

185.  Plasmodium  vivax,  an  atypical  macrogametocyte 767 

186.  Plasmodium  vivax 767 

187.  Plasmodium  vivax ' 768 

188.  Plasmodium  vivax 769 

189.  Plasmodium  malarise 770 

190.  Plasmodium  malarise 770 

191.  Plasmodium  malarise 771 

192.  Plasmodium  falciparum 771 

193.  Plasmodium  falciparum 772 

194.  Plasmodium  falciparum,  male  crescent 773 

195.  Comparison  of  culex  and  anopheles 779 

196.  Babesia  bigeminum 787 

197.  Texas  fever  tick  (Margaropus  annulatus) 789 

198.  Balantidium  coli  in  a  follicle  of  the  colon,  breaking  through  the  mucosa   .  793 


'     SECTION   I 

THE    GENERAL    BIOLOGY    OF   BACTERIA   AND    THE 
TECHNIQUE    OF    BACTERIOLOGICAL   STUDY 


CHAPTER   I 


THE  DEVELOPMENT   AND   SCOPE   OF   BACTERIOLOGY 

As  we  trace  back  to  their  ultimate  origins  the  lines  of  development 
of  living  beings  of  the  animal  and  plant  kingdoms,  we  find  them  con- 
verging toward  a  common  type,  represented  by  a  large  group  of  uni- 
cellular organisms,  so  simple  in  structure,  so  unspecialized  in  function, 
that  their  classification  in  either  the  realm  of  plants  or  that  of  animals 
becomes  little  more  than  an  academic  question.  However,  even  such 
microorganisms,  in  which  the  functions  of  nutrition,  respiration,  loco- 
motion, and  reproduction  are  concentrated  within  the  confines  of  a 
single  cell,  and  in  which  adaptation  to  special  conditions  more  readily 
brings  about  modifications  leading  to  the  production  of  a  multitude  of 
delicately  graded  transitional  forms,  fall  into  groups  which,  either  in 
structure  or  in  biological  attributes  show  evidence  of  a  tendency 
toward  one  or  the  other  of  the  great  kingdoms. 

Most  important  of  these  unicellular  forms,  for  the  student  of  medical 
science,  are  the  bacteria  and  the  protozoa. 

The  former,  by  reason  of  their  undifferentiated  protoplasm,  their 
occasional  possession  of  cellulose  membranes,  their  biological  tendency 
to  synthetize,  as  well  as  to  break  down  organic  compounds,  and  because 
of  the  transitional  forms  which  seem  to  connect  them  directly  with  the 
lower  plants,  are  generally  placed  in  the  plant  kingdom.  The  latter, 
chiefly  on  the  basis  of  metabolism,  are  classified  with  the  animals. 

Knowledge  of  the  existence  of  microorganisms  as  minute  as  the 
ones  under  discussion,  was  of  necessity  forced  to  await  the  perfection  of 
instruments  of  magnification.  It  was  not  until  the  latter  half  of  the 
seventeenth  century,  therefore,  that  the  Jesuit,  Kircher,  in  1659,  and 
the  Dutch  linen-draper,  van  Leeuwenhoek,  in  1675,  actually  saw  and 

1 


2  BIOLOGY  AND  TECHNIQUE 

described  living  beings  too  small  to  be  seen  with  the  naked  eye.  There 
can  be  no  doubt  that  the  small  bodies  se^n^hy  these  men  and  their  many 
immediate  successors  were,  at  lea^st In  part,  bacteria..  And  indeed  the 
descriptions  and  illustrations  of  several  of  the  earliest  workers  cor- 
respond with  many  oM^he  forms  which  are  well  known  to  us  at  the 
present  day. 

During  the  century  following  the  work  of  these  pioneers,  the  efforts 
of  investigators  lay  chiefly  in  the  more  exact-morphological  description 
of  some  of  the  forms  of  unicellular  life,  already  known.  Conspicuous 
among  the  work  of  this  period  is  that  of  Otto  Friedrich  Miiller.  In  the 
generation  following  MuUer's  work,  however,  a  marked  advance  in  the 
study  of  these  forms  was  made  by  Ehrenberg,*  who  established  a 
classification  which,  in  some  of  its  cardinal  divisions,  is  retained  until 
the  present  day. 

Meanwhile  the  regularity  with  which  these  "animalcula"  or  "in- 
fusion animaicula"  were  demonstrable  in  tartar  from  the  teeth,  in  intes- 
tinal contents,  in  well-water,  etc.,  had  begun  to  arouse  in  the  minds  of 
the  more  advanced  physicians  of  the  time  a  suspicion  as  to  a  possible 
relationship  of  these  minute  forms  with  disease.  The  conception  of 
"contagion,''  or  transmission  of  a  disease  from  one  human  being  to 
another,  was,  however,  even  at  this  time,  centuries  old.  The  fact  had 
been  recognized  by  Aristotle,  had  been  reiterated  by  medieval  philos- 
ophers, and  had  led,  in  1546,  to  the  division  of  contagious  diseases  by 
Fracastor,  into  those  transmitted  "per  contactum,''  and  those  con- 
veyed indirectly  "per  fomitem.''  It  was  for  these  mysterious  facts  of 
the  transmissibility  of  disease,  that  clinicians  of  the  eighteenth  centurj'-, 
with  remarkable  insight,  saw  an  explanation  in  the  microorganisms  dis- 
covered by  Leeuwenhoek  and  his  followers. 

In  fact,  Plenciz  of  Vienna,  writing  in  1762,  not  only  expressed 
a  belief  in  the  direct  etiological  connection  between  microorganisms 
and  some  diseases,  but  was  the  first  to  advance  the  opinion  that  each 
malady  had  its  own  specific  causal  agent,  which  multiplied  enormously 
in  the  diseased  body.  The  opinions  of  this  author,  if  translated  into 
the  language  of  our  modern  knowledge  of  the  subject,  came  remark- 
ably near  to  the  truth,  not  only  as  regards  etiology  and  transmission, 
but  also  in  their  suggestion  of  a  specific  therapy  for  each  disease. 

The  conception  of  a  "  contagium  vivum ''  was  thus  practically  es- 
tablished with  the  work  of  Plenciz  and  many  others  who  followed  in 

1 "  Die  Infusionstierchen/'  etc.,  Leipzig.  1838.  • 


DEVELOPMENT   AND   SCOPE   OF   BACTERIOLOGY  3 

his  train,  but  the  astonishingly  shallow  impression  which  the  acute 
reasoning  of  these  men  left  upon  the  medical  thought  of  their  day 
furnishes  an  excellent  example  of  the  futility  of  the  most  penetrating 
speculation  when  unsupported  by  experimental  data. 

The  real  advancement  in  the  scientific  development  of  the  subject 
was  destined  to  be  carried  on  along  entirely  different  lines.  In  1837, 
Schwann,  a  botanist,  showed  that  the  yeasts,  found  in  fermenting  sub- 
stances, were  Hving  beings,  which  bore  a  causal  relationship  to  the  proc- 
ess of  fermentation.  At  almost  the  same  time,  similar  observations  were 
made  by  a  French  physicist,  Cagniard-Latour.  The  opinions  advanced 
by  these  men  on  the  nature  of  fermentation  aroused  much  interest 
and  discussion,  since,  at  that  time  and  for  a  long  period  thereafter, 
fermentation  was  ascribed  universally  to  proteid  decomposition,  a  process 
which  was  entirely  obscure  but  firmly  believed  to  be  of  a  purely  chemical 
nature. 

Although  belief  in  the  discovery  of  Schwann  did  not  completely 
master  the  field  until  after  Pasteur  had  completed  his  classical  studies 
upon  the  fermentations  occurring  in  beer  and  wine,  yet  the  conception 
of  a  "  fermentum  vivum  "  aroused  much  speculation,  and  the  attention 
of  physicians  and  scientists  was  attracted  to  the  many  analogies  ex- 
isting between  phenomena  of  fermentation  and  those  of  disease. 

The  conception  of  such  an  analogy,  however,  was  not  a  new  thought 
in  the  philosophy  of  the  time.  Long  before  Schwann  and  Cagniard- 
Latour,  the  philosopher  Robert  Boyle,  working  in  the  seventeenth 
century,  had  prophesied  that  the  mystery  of  infectious  diseases  would  be 
solved  by  him  who  should  succeed  in  elucidating  the  nature  of  fermenta- 
tion. 

Nevertheless,  the  diligent  search  for  microorganisms  in  relation  to 
various  diseases  which  followed,  led  to  few  results,  and  the  successes 
which  were  attained  were  limited  to  the  diseases  caused  by  some  of 
the  larger  fungi,  favus  (1839),  thrush  (1839),  and  pityriasis  versicolor 
(1846).  During  this  time  of  ardent  but  often  poorly  controlled  etiolog- 
ical research,  it  was  Henle  who  formulated  the  postulates  of  conserva- 
tism, almost  as  rigid  as  the  later  postulates  of  Koch,  requiring  that 
proof  of  the  etiological  relationship  of  a  microorganism  to  a  disease 
could  not  be  brought  merely  by  finding  it  in  a  lesion  of  the  disease,  but 
that  constant  presence  in  such  lesions  must  be  proven  and  isolation  and 
study  of  the  microorganism  away  from  the  diseased  body  must  be  car- 
ried out. 

It  was  during  this  period  also  that  one  of  the  most  fundamental 


4  BIOLOGY  AND/TECHNIQUEX 

questions,  namely,  that  of  the  origin  of  these  minlute  living  beings,  wa^ 
being  discussed  with  much  passion  by  the  scientific  world.  It  was-held 
by  the  conservative  majority  that  the  microorganisms  described  by 
Leeuwenhoek  and  others  after  him,  were  produced  by  spontaneous 
generation.  The  doctrine  of  spontaneous  generation,  in  fact,  was 
solidly  established  and  sanctified  by  tradition,  and  had  been  applied 
in  the  past  not  alone  to  microorganisms.*  And  it  must  not  be  forgotten 
that  without  the  aid  of  our  modern  methods  of  study,  satisfactory 
proof  for  or  against  such  a  process  was  not  easily  brought. 

Needham,  who  published  in  1749,  had  spent  much  time  in  fortify- 
ing his  opinions  in  favor  of  spontaneous  generation  by  extensive  ex- 
perimentation. He  had  placed  putrefying  material  and  vegetable  in- 
fusions in  sealed  flasks,  exposing  them  for  a  short  time  to  heat,  by 
immersing  them  in  a  vessel  of  boiling  water,  and  had  later  shown  them 
to  be  teeming  with  microorganisms.  He  was  supported  in  his  views 
by  no  less  an  authority  than  Buffon.  The  work  of  Needham,  however, 
showed  a  number  of  experimental  inaccuracies  which  were  thoroughly 
sifted  by  the  Abbe  Spallanazani.  This  investigator  repeated  the  ex- 
periments of  Needham,  employing,  however,  greater  care  fn  sealing  his 
flasks,  and  subjecting  them  to  a  more  thorough  exposure  to  heat. 
His  results  did  not  support  the  views  of  Needham,  but  were  answered 
by  the  latter  with  the  argument  that  by  excessive  heating  he  had  pro- 
duced chemical  changes  in  his  solutions  which  had  made  spontaneous 
generation  impossible. 

The  experiments  of  Schulze,  in  1836,  who  failed  to.  find  living  organ- 
isms in  infusions  which  had  been  boiled,  and  to  which  air  had  been 
admitted  only  after  passage  through  strongly  acid  solutions,  and  similar 
results  obtained  by  Schwann,  who  had  passed  the  air  through  highly 
heated  tubes,  were  open  to  criticism  by  their  opponents,  who  claimed 
that  chemical  alteration  of  the  air  subjected  to  such  drastic  influences, 
had  been  responsible  for  the  absence  of  bacteria  in  the  infusion.  Similar 
experiments  by  Schroeder  and  Dusch,  who  had  stoppered  their  flasks 
with  cotton  plugs,  were  not  open  to  this  objection,  but  had  also  failed  to 
convince.    The  question  was  not -definitely  settled  until  the  years  im- 

» Valleri-Radot,  in  his  life  of  Pasteur,  stated  that  Van  Helmont,  in  the  six- 
teenth century,  had  given  a  celebrated  prescription  for  the  creation  of  mice 
from  dirty  linen  and  a  few  grains  of  wheat  or  pieces  of  cheese.  During  the  centu- 
ries following,  although,  of  course,  such  remarkable  and  amusing  beliefs  no  longer 
held  sway,  nevertheless  the  question  of  spontaneous  generation  of  minute  and 
structureless  bodies,  like  the  bacteria,  still  foimd  learned  and  thoughtful  partisans. 


DEVELOPMENT    AND   SCOPE    OP   BACTERIOLOGY  5 

mediately  following  1860,  when  Pasteur  conducted  a  series  of  experi- 
ments which  were  not  only  important  in  incontrovertibly  refuting  the 
doctrine  of  spontaneous  generation,  but  in  establishing  the  principles 
of  scientific  investigation  which  have  influenced  bacteriological  re- 
search since  his  time.^ 

Pasteur  attacked  the  problem  from  two  points  of  view.  In  the 
first  place  he  demonstrated  that  when  air  was  filtered  through  cotton- 
wool, innumerable  microorganisms  were  deposited  upon  the  filter.  A 
single  shred  of  such  a  contaminated  filter  dropped  into  a  flask  of  pre- 
viously sterilized  nutritive  fluid,  sufiiced  to  bring  about  a  rapid  and 
luxuriant  growth  of  microorganisms.  In  the  second  place,  he  succeeded 
in  showing  that  similar,  sterilized  "  putrescible "  liquids,  if  left  in  con- 
tact with  air,  would  remain  uncontaminated  provided  that  the  en- 
trance of  dust  particles  were  prohibited.  This  he  succeeded  in  doing  by 
devising  flasks,  the  necks  of  which  had  been  drawn  out  into  fine  tubes 
bent  in  the  form  of  a  U.  The  ends  of  these  U-tubes,  being  left  open, 
permitted  the  sedimentation  of  dust  from  the  air  as  far  as  the  lowest 
angle  of  the  tube,  but,  in  the  absence  of  an  air  current,  no  lust  was 
carried  up  the  second  arm  into  the  liquid.  In  such  flasks  he  showed 
that  no  contamination  took  place  but  could  be  immediately  induced 
by  slanting  the  entire  apparatus  until  the  liquid  was  allowed  to  run 
into  the  bent  arm  of  the  U-tube.  Finally,  by  exposing  a  series  of 
flasks  containing  sterile  yeast  infusion,  at  different  atmospheric  levels, 
in  places  in  which  the  air  was  subject  to  varying  degrees  of  dust  con- 
tamination, he  showed  an  inverse  relationship  between  the  purity  ox 
the  air  and  the  contaminatioti  of  his  flasks  with  microorganisms. 

The  doctrine  of  spontaneous  generation  had  thus  received  its  final 
refutation,  except  in  one  particular.  It  was  not  yet  clear  why  com- 
plete sterility  was  not  always  obtained  by  the  application  of  definite 
degrees  of  heat.  This  final  link  in  the  chain  of  evidence  was  supplied, 
some  ten  years  later,  by  Cohn,  who,  in  1871,  was  the  first  to  observe  and 
correctly  interpret  bacterial  spores  and  to  demonstrate  their  high  powers 
of  resistance  against  heat  and  other  deleterious  influences. 

^  In  a  letter  to  his  foremost  opponent,  at  this  period,  Pasteur  writes:  "In 
experimental  science,  it  is  always  a  mistake  not  to  doubt  when  facts  do  not  compel 
affirmation." 

The  critical  spirit  pervading  the  scientific  thought  of  that  time  in  France  ii^ 
also  well  expressed  by  Oliver  Wendell  Holmes,  who  said  that  he  had  learned  three 
things  in  Paris:  "Not  to  take  authority  when  I  can  have  facts,  not  to  guess  when 
I  can  know,  and  not  to  think  that  a  man  must  take  physic  because  he  is  sick." 


6  BIOLOGJV  AND  TECHNIQUE 

Meanwhile,  Pasteur,  parallel  with  his  researches  upon  spontaneous 
generation,  had  been  ^eirrying  on  experiments  upon  the  subject  of 
fermentation  along  t!ie  lines  suggested  by  Cagniard-Latour.  As  a 
consequence  of  these  experiments,  he  not  only  confirmed  the  opinions 
both  of  this  author  and  of  Schwann  concerning  the  fermentation  of  beer 
and  wine  by. yeasts,  but  was  able  to  show  that  a  number  of  o^her  fer- 
mentations, such  as  those  of  lactic  and  butyric  acid,  as  well  as  the  de- 
composition of  organic  matter  by  putrefaction,  were  directly  due  to  the 
action  of  microorganisms.  It  was  the  discovery  of  the  living  agents 
underlying  putrefaction,  especially,  which  exerted  the  most  active 
influence  upon  the  medical  research  of  the  day.  This  is  illustrated  by 
Lister's  work.  The  suppurative  processes  occurring  in  infected  wounds 
had  long  been  regarded  as  a  species  of  putrefaction,  and  Lord  Lister, 
working  directly  upon  the  premises  supplied  by  Pasteur,  introduced 
into  both  the  active  and  prophylactic  treatment  of  surgical  wounds, 
the  antiseptic  principles  which  alone  have  made  modern  surgery  possible. 

There  now  followed  a  period  in  which  bacteriological  investigation 
was  concentrated  upon  problems  of  etiology.  Stimulated  by  Pasteur's 
successes,  the  long-cherished  hope  of  finding  some  specific  microorgan- 
ism as  the  causal  agent  in  each  infectious  disease  was  revived. 

PoUender,  in  1855,  had  reported  the  presence  of  rod-shaped  bodies 
in  the  blood  and  spleen  of  animals  dead  of  anthrax.  Brauell,  several 
years  later,  had  made  sijnilar  observations  and  had  expressed  definite 
opinions  as  to  the  causative  relationship  of  these  rods  to  the  disease. 
Convincing  proof,  however,  had  not  been  brought,  by  either  of  these 
observers.  Finally,  in  1863,  Davaine,  in'  a  series  of  brilliant  investi- 
gations, not  only  confirmed  the  observations  of  the  two  authors  men- 
tioned above,  but  succeeded  in  demonstrating  that  the  disease  could 
be  transmitted  by  means  of  blood  containing  these  rods  and  could  never 
be  transmitted  by  blood  from  which  these  rods  were  absent.  Anthrax, 
thus,  is  the  first  disease  in  which  definite  proof  of  bacterial  causation 
was  brought. 

Speaking  before  the  French  Academy  of  Medicine  at  this  time, 
Davaine  suggested  that  the  manifestations  of  the  disease  might  in 
reality  represent  the  results  of  a  specific  fermentation  produced  by  the 
bacilli  he  had  found.  This,  in  a  crude  way,  expresses  the  modern 
conception  of  infectious  disease. 

Within  a  f^  years  after  this,  1868,  the  adherents  of  the  parasitic 
theory  of  infectious  diseases  were  further  encouraged  by  the  discovery, 
by  Obermeier,  of  a  spirillum  in  the  blood  of  patients  suffering  from 


DEVELOPMENT  AND  SCOPE  OF  BACTERIOLOGY  7 

relapsing  fever.  It  is  not  surprising  that  the  successes  attained  in  these 
diseases,  fostering  hope  of  analogous  results  in  all  other  similar  condi- 
tions, but  without  the  aid  of  adequate  experimental  methods,  should 
have  led  to  many  unjustified  claims  and  to  much  fantastic  theorizing. 
Thus  Hallier,  at  about  this  time,  advanced  a  theory  as  to  the  etiology 
of  infectious  diseases,  in  which  he  attributed  all  such  conditions  to  the 
moulds  or  hyphomycetes,  regarding  the  smaller  form  or  bacteria  as 
developmental  stages  of  these  more  complicated  forms.  Extravagant 
conjectures  of  this  kind,  however,  did  not  maintain  themselves  for  any 
length  of  time  in  the  light  of  the  critical  attitude  which  was.  already 
pervading  bacteriological  research. 

Progress  was  made  during  the  years  immediately  following,  chiefly 
in  the  elucidation  of  suppurative  processes.  Rindfleisch,  von  Reckling- 
hausen, and  Waldeyer,  almost  simultaneously,  described  bodies  which 
they  observed  in  sections  of  tissue  containing  abscesses,  and  which  they 
believed  to  be  microorganisms.  Notable  support  was  given  to  their 
opinion  by  similar  observations  made  upon  pus  by  Klebs,  in  1870.  In 
view,  however,  of  the  purely  morphological  nature  of  their  studies,  the 
opinions  of  these  observers  did  not  entirely  prevail.  Satisfactory 
methods  of  cultivation  and  isolation  had  not  yet  been  developed,  and 
Billroth  and  his  followers,  with  a  conservatism  entirely  justified  under 
existing  conditions,  while  admitting  the  constant  presence  of  bacteria 
in  purulent  lesions,  denied  their  etiological  significance.  The  contro- 
versy that  followed  was  rich  in  suggestions  which  greatly  facilitated 
the  work  of  later  investigators,  but  could  not  be  definitely  settled  until 
1880,  when  Koch  introduced  the  technical  methods  which  have  made 
bacteriology  an  exact  science.  By  the  use  of  solid  nutritive  media,  the 
isolation  of  bacteria  and  their  biological  study  in  pure  culture  were  made 
possible.  At  about  the  same  time  the  use  of  anilin  dyes,  developed 
by  Weigert,  Koch,  and  Ehrlich,  was  introduced  *jito  morphological  study 
and  facilitated  the  observation  of  the  finer  structural  details  which  had 
been  unnoticed  while  only  the  grosser  methods  employed  for  tissue 
staining  had  been  available. 

With  the  publication  of  Koch's  work,  there  began  an  era  unusually 
rich  in  results  held  in  leash  heretofore  by  inadequate  technical  methods. 
The  discovery  of  the  typhoid  bacillus  in  1880,  of  the  bacillus  of  fowl 
cholera  and  the  pneumococcus  in  the  same  year,  and  of  the  tubercle 
bacillus  in  1882,  initiated  a  series  of  etiological  discoveries  which,  ex- 
tending over  not  more  than  fifteen  years,  elucidated  the  causation 
of  a  majority  of  the  infectious  diseases. 


8  BIOLOGY  AND  TECHNIQUE 

Coincident  with  the  elucidation  of  etiological  facts  began  the  inquiry 
into  that  field  which  is  now  spoken  of  as  the  science  of  immunity.  The 
phenomena  which  accompany  the  development  of  insusceptibility 
to  bacterial  infections  in  man  and  in  animals,  first  studied  by  Pasteur, 
have  become  the  subject  of  innumerable  researches  and  have  led  to 
results  of  the  utmost  practical  value. 

The  problems  which  were  encountered  were  firgit>  studied  from  a 
purely  bacteriological  point  of  view,  but  their  solution  has  shed  light 
upon  biological  principles  of  the  broadest  application.  Investigations 
into  the  properties  of  immune  sera,  while  making  bacteriology  one  of 
the  most  important  branches  of  diagnostic  and  therapeutic  medicine, 
have,  at  the  same  time,  inseparably  linked  it  with  physiology  and 
experimental  pathology. 

By  the  revelations  of  etiological  research,  and  by  the  study  of  the 
biological  properties  of  pathogenic  bacteria,  contagion,  an  enemy  hitherto 
unseen  and  mysterious,  was  unmasked,  and  rational  campaigns  of  public 
sanitation  and  personal  hygiene  were  made  possible.  Upon  the  same 
elucidations  has  depended  the  development  of  modern  surgery — a 
science  which  without  asepsis  and  antisepsis  would  have  been  doomed 
to  remain  in  its  medieval  condition. 

Apart  from  its  importance  in  the  purely  medical  sciences,  the  study 
of  the  bacteria  has  shed  beneficial  light,  moreover,  upon  many  other 
fields  of  human  activity.  In  their  relationship  to  decomposition,  the 
conditions  of  the  soil,  and  to  diseases  of  plants,  the  bacteria  Jiave  been 
found  to  occupy  a  position  of  great  importance  in  agriculture.  Knowl- 
edge of  bacterial  and  yeast  ferments,  furthermore,  has  become  the  scien- 
tific basis  of  many  industries,  chiefly  those  concerned  in  the  production 
of  wine,  beer,  and  dairy  products. 

The  scope  of  bacteriology  is  thus  a  wide  one,  and  none  of  its  various 
fields  has,  as  yet,  been  fully  explored.  The  future  of  the  science  is  rich 
in  allurement  of  interest,  in  promise  of  result,  and  in  possible  benefit 
to  mankind. 


CHAPTER  II 

GENERAL   MORPHOLOGY,    REPRODUCTION,    AND   CHEMICAL 
AND  PHYSICAL  PROPERTIES  OF   THE   BACTERIA 

Bacteria  are  exceedingly  minute  unicellular  organisms  which  maj 
occur  perfectly  free  and  singular,  or  in  larger  or  smaller  aggregations, 
thus  forming  multicellular  groups  or  colonies,  the  individuals  of  which 
are,  however,  physiologically  independent. 

The  cells  themselves  have  a  number  of  basic  or  ground  shapes  which 
may  be  roughly  considered  in  three  main  classes:  The  cocci  or  spheres, 
the  bacilli  or  straight  rods,  and  the  spirilla  or  curved  rod  forms. 

The  cocci  are,  when  fully  developed  and  free,  perfectly  spherical. 
When  two  or  more  are  in  apposition,  they  may  be  slightly  flattened  along 
the  tangential  surfaces,  giving  an  oval  appearance. 

The  bacilli,  or  rod-shaped  forms,  consist  of  elongated  cells  whose 
long  diameter  may  be  from  two  to  ten  times  as  great  as  their  width, 
with  ends  squarely  cut  off,  as  in  the  case  of  bacillus  anthracis,  or  gently 
rounded  as  in  the  case  of  the  typhoid  bacillus. 

The  spirilla  may  vary  from  small  comma-shaped  microorganisms, 
containing  but  a  single  curve,  to  longer  or  more  sinuous  forms  which 
may  roughly  be  compared  to  a  corkscrew,  being  made  up  of  five,  six, 
or  more  curves.  The  turns  in  the  typical  microorganisms  of  this  class  are 
always  in  three  planes  and  are  spiral  rather  than  simply  curved. 

Among  the  known  microorganisms,  the  bacilli  by  far  outnumber 
other  forms,  and  are  probably  the  most  common  variety  of  bacteria  in 
existence.  Many  variations  from  these  fundamental  types  may  occur 
even  under  normal  conditions,  but  contrary  to  earlier  opinions  it  is 
now  positively  known  that  cocci  regularly  reproduce  cocci,  bacilli 
bacilli,  and  spirilla  spirilla,  there  being,  as  far  as  we  know,  no  mutation 
from  one  form  into  another. 

The  size  of  bacteria  is  subject  to  considerable  variation.  Cocci  may 
vary  from  .15  /jl  to  2.  /x  in  diameter.  The  average  size  of  the  ordinary 
pus  coccus  varies  from  .8  n  to  1.2  /z  in  diameter.  Fischer  has  given  a 
graphic  illustration  of  the  size  of  a  staphylococcus  by  calculating  that 
one  billion  micrococci  could  easily  be  contained  in  a  drop  of  water  hav- 

9 


10  BIOLOGY  And  technique 

ing  a  volume  of  one  cubic  millimeter.  Among  the  bacilli  the  range  of 
size  is  subject  to  even  greater  variations.  Probably  the  smallest  of  the 
common  bacilli  is  the  bacillus  of  influenza  which  measures  about  .5  /i  in 
length  by  .2  j«  in  thickness.  The  limit  of  the  optical  possibilities  of  the 
modern  microscope  is  almost  reached  by  some  of  the  known  micro- 
organisms, and  it  is  not  at  all  out  of  question  that  some  of  the  diseases, 
for  which,  up  to  the  present  time,  no  specific  microorganisms  have 


Fig.  1. — ^Types  of  Bacterial  Morphology. 

been  found,  may  be  caused  by  bacteria  so  small  as  to  be  invisible  by  any 
of  our  present  methods.  In  fact,  the  virus  causing  the  peripneumonia  of 
cattle  has  been  shown  to  pass  through  the  pores  of  a  Berkefeld  filter, 
which  are  impenetrable  to  the  smallest  of  the  known  bacteria.* 


MORPHOLOGY   OF   THE   BACTERIAL   CELL 

When  unstained,  most  bacteria  are  transparent,  colorless,  and  ap- 
parently homogeneous  bodies  with  a  low  refractive  index.  The  cells 
themselves  consist  of  a  mass  of  protoplasm,  surrounded,  in  most  in- 
stances, by  a  delicate  cell  membrane. 

The  presence  of  a  nucleus^  in  bacterial  cells,  though  denied  by  the 
earlier  writers,  has  been  demonstrated  beyond  question  by  Zettnow, 
Nakanishi,^  and  others.  The  original  opinion  of  Zettnow  was  that  the 
entire  bacterial  body  consisted  of  nuclear  material  intimately  inter- 
mingled with  the  Cytoplasm.    The  opinion  now  held  by  most  observers 

'  Nocard  and  Roux,  Ann.  Past.,  12,  1898. 

2  A.  Fischer,  Jahrbiicher  f.  wissen.  Botanik,  xxvii, 

•  Ndkanishi,  Miinch.  med.  Woch.,  vi,  1900. 


MORPHOLOGY,  REPRODUCTION,  ETC.  11 

who  have  studied  this  phase  of  the  subject  favors  the  existence  of  an 
ectoplasmic  zone  which  includes  cell  membrane  and  fiagella,  but  is 
definitely  a  part  of  the  cytoplasm,  and  an  entoplasm  in  which  is  con- 
centrated the  nuclear  material.  Biitschli '  claims  to  have  demonstrated 
within  this  entoplasmic  substance  a  reticular  meshwork,  between  the 
spaces  of  which  lie  granules  of  chromophilic  or  nuclear  material. 
Confirmation  of  this  opinion  has  been  brought  by  Zettnow"  and  others. 
Nakanishi,  working  with  a  special  staining  method,  asserts  that  some 
microorganisms  show  within  the  entoplasmic  zone  a  well-defined, 
minute,  round  or  oval  nucleus,  which  possesses  a  definitely  charac- 
teristic staining  reaction.^ 

In  the  bodies  of  a  large  number  of  bacteria,  notably  in  those  of  the 
diphtheria  group,  Ernst,*  Babes,^  and  others  have  demonstrated 
granular,  deeply  staining  bodies  now  spoken  of  as  metachromatic  granules, 
or  Babes-Ernst  granules,  or,  because  of  their  frequent  position  at  the  ends 
of  bacilli,  as  polar  bodies.  These  structures  are  irregular  in  size  and 
number,  and  have  a  strong  affinity  for  dyes.  They  are  stained  dis- 
tinctly dark  in  contrast  to  the  rest  of  the  bacterial  cell  with  methylene 
blue,  and  may  be  demonstrated  by  the  special  methods  of  Neisser  and 
of  Roux.^  Their  interpretation  has  been  a  matter  of  much  difficulty 
and  of  varied  opinion.  Those  who  first  observed  them  held  that  they 
were  a  part  of  the  nuclear  material  of  the  cell.  Others  have  regarded 
them  as  an  early  stage  in  spore  formation,  or  as  arthrospores.'  Again, 
they  have  been  interpreted  as  structures  comparable  to  the  centrosomes 
of  other  unicellular  forms.  As  a  matter  of  fact,  the  true  nature  of  these 
bodies  is  by  no  means  certain.  They  are  present  most  regularly  in 
microorganisms  taken  from  young  ^nd  vigorous  cultures  or  in  those 
taken  directly  from  the  lesions  of  disease.    It  is  unlikely  that  they  repre- 


»  Biitschli, "  Bau  der  Bakterien,"  Leipzig,  1890.    2  Zettnow,  Zeit.  f .  Hyg.,  xxiv,  1897. 

3  The  method  of  Nakanishi  is  carried  out  as  follows:  Thoroughly  cleansed 
slides  are  covered  with  a  saturated  aqueous  solution  of  methylene  blue.  This  is 
spread  over  the  slide  in  an  even  film  and  allowed  to  dry.  After  drying,  the  slide 
should  be  of  a  transparent,  sky-blue  color.  The  microorganisms  to  be  examined  are 
then  emulsified  in  warm  water,  or  are  taken  from  the  fluid  media,  and  dropped  upon 
a  cover  slip.  This  is  placed,  face  downward,  upon  the  blue  ground  of  the  slide.  In 
this  way,  bacteria  are  stained  without  fixation.  Nakanishi  claims  that  by  this 
method  the  entoplasm  is  stained  blue,  while  the  nuclear  material  appears  of  a  reddish 
or  purplish  hue. 

'»  Ernst,  Zeit.  f.  Hyg.,  iv,  1888.  » Babes,  Zeit.  f.  Hyg.,  v,  1889. 

•  See  section  on  stains,  p.  107.  »  See  section  on  sporulation,  p.  16. 


12 


BIOLOGY  AiND  TECHNIQUE 


sent  structures  in  any  way  comparable  to  spores,  since  cultures  con- 
taining individuals  showing  metachromatic  granules  are  not  more 
resistant  to  deleterious  influences  than  are  others.  Their  abundant 
presence  in  young  vigorous  cultures  may  indicate  a  relationship  between 
them  and  the  growth  energy  of  the  microorganisms.  There  is  no  proof, 
however,  that  these  bodies  affect  the  virulence  of  the  bacteria. 

Cell  Membrane  and  Capsule. — Actual  proof  of  the  existence  of  a  cell 
membrane  has  been  brought  in  the  cases  of  some  of  the  larger  forms 
only,^  but  the  presence  of  such  envelopes  may  be  inferred  for  most 

bacteria  by  their  behavior  during 


'*A 

m  *■ 


t 


♦  1 


# 


•*' 


Fig.  2. — ^Bacterial  Capsules, 


plasmolysis,  where  definite  retrac- 
tion of  the  protoplasm  from  a 
well-defined  cell  outline  has  been 
repeatedly  observed.  The  occur- 
rence, furthermore,  of  so-called 
"shadow  forms"  which  appear  as 
empty  capsules,  and  of,  occasion- 
ally, a  well-outlined  cell  body, 
after  the  vegetative  form  has  en- 
tirely degenerated  in  the  course 
of  sporulation,  make  the  assump- 
tion of  the  presence  of  a  cell 
membrane  appear  extremely  well 
founded.  Differing  from  the  cell 
membranes  of  plant  cells,  cellulose 
has  not,  except  in  isolated  instances,  been  demonstrable  for  bacteria, 
and  the  membrane  is  possibly  to  be  regarded  rather  as  a  peripheral 
protoplasmic  zone,  which  remains  unstained  by  the  usual  manipula- 
tions. Zettnow,^  who  has  carefully  studied  the  structure  of  some  of 
the  larger  forms,  takes  the  latter  view,  and  regards  the  "  ectoplasmic " 
zone  as  a  part  of  the  cell  protoplasm  devoid  of  nuclear  material.  Zett- 
now's  opinion  is  borne  out  by  the  greatly  increased  size  of  the  bacterial 
cells  as  seen  by  means  of  special  stains. 

Many  bacteria  have  been  shown  to  possess  a  mucoid  or  gelatinous 
envelope  or  capsule.  According  to  Migula,^  such  an  envelope  is  present 
on  all  bacteria,  though  it  is  in  only  a  few  species  that  it  is  sufficiently 
well  developed  and  stable  to  be  easily  demonstrable  and  of  differential 


*  Biitschli,  loc.  cit.  ^  Zettnow,  loc.  cit. 

3  Migulaf  "Systeme  d.  Bakterien,"  1,  p.  56. 


MORPHOLOGY,  REPRODUCTION,  ETC.  13 

value.  When  stained,  the  capsule  takes  the  ordinary  anilin  dyes  less 
deeply  than  does  the  bacterial  cell  body,  and  varies  greatly  in  thickness, 
ranging  from  a  thin,  just  visible  margin  to  dimensions  four  or  five 
times  exceeding  the  actual  size  of  the  bacterial  body  itself.  This  struc- 
ture is  perfectly  developed  in  a  limited  number  of  bacteria  only  in  which 
it  then  becomes  an  important  aid  to  identification.  Most  prominent 
among  such  bacteria  are  Diplococcus  pneumoniae.  Micrococcus  tetra- 
genus,  the  bacilli  of  the  Friedlander  group,  and  B.  aerogenes  capsulatus. 
The  development  of  the  capsule  seems  to  depend  intimately  upon  the 
environment  from  which  the  baqjeria  are  taken.  It  is  most  easily  de- 
monstrable in  preparations  of  bacteria  taken  directly  from  animal  tis- 
sues and  fluids,  or  from  media  containing  animal  serum  or  milk.  If 
cultivated  for  a  prolonged  period  upon  artificial  media,  many  otherwise 
capsulated  microorganisms  no  longer  show  this  characteristic  structure. 

Capsules  may  be  demonstrated  on  bacteria  taken  from  artificial 
media  most  successfully  when  albuminous  substances,  such  as  ascitic 
fluid  or  blood  serum,  are  present  in  the  culture  media,  or  when  the 
bacteria  are  smeared  upon  cover  slip  or  slide  in  a  drop  of  beef  or  other 
serum.^  Most  observers  believe  that  the  capsule  represents  a  swelling 
of  the  ectoplasmic  zone  of  bacteria.  By  others  it  is  regarded  as  an 
evidence  of  the  formation  of  a  mucoid  intercellular  substance,  some  of 
which  remains  adherent  to  the  individual  bacteria  when  removed  from 
cultures.  It  is  noticeable,  indeed,  that  some  of  the  capsulated  bacteria, 
especially  Streptococcus  mucosus  and  B.  mucosus  capsulatus,  develop 
such  slimy  and  gelatinous  colonies  that,  when  these  are  touched  with  a 
platinum  wire,  mucoid  threads  and  strings  adhere  to  the  loop.  Exactly 
what  the  significance  of  the  capsules  is  cannot  yet  be  decided. 

There  is,  however,  definite  reason  to  believe  that  there  is  a  direct 
relation  between  virulence  and  capsulation;  capsulated  bacteria  are 
less  easily  taken  up  by  phagocytes  than  are  the  non-capsulated  mem- 
bers of  the  same  species.  Also,  as  Forges  and  others  have  shown, 
capsulated  organisms  are  not  easily  amenable  to  the  agglutinating  action 
of  immune  sera.  Many  bacteria  (plague,  anthrax)  which  are  habitu- 
ally uncapsulated  on  artificial  media  acquire  capsules  within  the  in- 
fected animal  body.  Also  in  some  species  (pneumococci),  the  loss 
of  capsule  formation  as  cultivated  on  the  simpler  media  is  accompanied 
by  a  diminution  of  virulence. 

Organs  of  Locomotion. — When  suspended  in  a  drop  of  fluid  many 
bacteria  are  seen  to  be  actively  motile.     It  is  important,  however,  in 

*  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


14  BIOLOGY  A\NrD  TECHNIQUE 

all  cases  to  distinguish  between  actual  motility  and  the  so-called  Brown- 
ian  or  molecular  movement  which  takes  place  whenever  small  particles 
are  held  in  suspension  in  a  fluid. 

Brownian  or  molecular  movement  is  a  phenomenon  entirely  ex- 
plained by  the  physical  principles  of  surface  tension,  and  has  absolutely 
no  relation  to  independent  motility.  It  may  be  seen  when  particles  of 
carmine  or  any  other  insoluble  substance  are  suspended  in  water,  and 
consists  in  a  rapid  to  and  fro  vacillation  during  which  there  is  actually 
no  permanent  change  in  position  of  the  moving  particle  except  inas- 
much as  this  is  influenced  by  current^in  the  drop. 

The  true  motility  of  bacteria,  on  the  other  hand,  is  active  motion 
due  to  impulses  originating  in  the  bacteria  themselves,  where  the  actual 
position  of  the  bacterium  in  the  field  is  permanently  changed. 

The  ability  to  move  in  this  way  is,  so  far  as  we  know,  limited  almost 
entirely  to  the  bacilli  and  spirilla,  there  being  but  few  instances  where 
members  of  the  coccus  group  show  active  motility.  In  all  cases,  with 
the  exception  of  some  of  the  spirochetes,  where  motility  may  occasionally 
be  due  to  an  undulating  membrane  marginally  placed  along  the  body, 
bacterial  motility  is  due  to  hair-like  organs  known  as  flagella.  These 
flagella  have  rarely  been  seen  during  life,  and  their  recognition  and  study 
has  been  made  possible  only  by  special  staining  methods,  such  as  those 
devised  by  Loeffler,  van  Ermengem,  Pitt,  and  others. 

In  such  stained  preparations,  the  bacterial  cell  bodies  often  appear 
thicker  than  when  ordinary  dyes  are  used,  and  the  flagella  apparently 
are  seen  to  arise  from  the  thickened  ectoplasmic  zone. 

The  flagella  are  long  filaments,  averaging  in  thickness  from  one-tenth 
to  one-thirtieth  that  of  the  bacterial  body,  which  often  are  delicately 
v/aved  and  undulating,  and,  judging  from  the  positions  in  which  they 
become  fixed  in  preparations,  move  by  a  wavy  or  screw-like  motion. 
In  length  they  are  subject  to  much  variation,  but  are  supposed  to  be 
generally  longer  in  old  than  in  young  cultures.  Very  short  flagella  have 
been  described  only  on  nitrosomonas,  one  of  the  nitrifying  bacteria 
discovered  by  Winogradsky.^ 

As  to  the  finer  structures  of  flagella,  little  can  be  made  out  except 
that  they  possess  a  higher  refractive  index  than  the  cell  body  itself, 
and  that  they  can  be  stained  only  with  those  dyes  which  bring 
clearly  into  view  the  supposedly  true  cytoplasm  of  the  cell. 
Whether  they  penetrate  this  cytoplasmic  membrane  or  whether  they 

1  Winogradsky,  Arch,  des  sci.  biologiques,  St.  Petersburg,  1892, 1,  1  and  % 


MORPHOLOGY,  REPRODUCTION,  ETC.  15 

are  a  direct    continuation  of    this  peripheral   zone   of  the   bacterial 
body,  can  not  be  decided. 

The  manner  in  which  bacteria  move  is  naturally  subject  to  some  var- 
iation depending  upon  the  number  and  position  of  the  flagella  possessed 
by  them.  Whether  bacteria  exercise  or  not  the  power  of  motility  de- 
pends to  a  large  extent  upon  their  present  or  previous  environment. 
They  are  usually  most  motile  in  vigorous  young  cultures  of  from  twenty- 
four  to  forty-eight  hours'  growth  in  favorable  media.  In  old  cultures 
motility  may  be  diminished  or  even  inhibited  by  acid  formation  or  by 
other  deleterious  products  of  the  bacterial  metabolism. 

At  the  optimum  growth-temperature  motility  is  most  active,  and  a 
diminution  or  increase  of  the  temperature 
to  any  considerable  degree  diminishes  or 
inhibits  it.  Thus  actively  motile  organisms, 
in  the  fluid  drop,  may  be  seen  to  diminish 
distinctly  in  activity  when  left  for  any 
prolonged  time  in  a  cold  room,  or  when  ^^^  3.-Arrangement  op 
the  preparation  is  chilled.    Any  influence.  Bacterial  Flagella. 

in  other  words,  chemical  or  physical,  which 

tends  to  injure  or  depress  physiologically  the  bacteria  in  any  way,  at 
the  same  time  tends  to  inhibit  their  motility. 

Messea^  has  proposed  a  classification  of  bacteria  which  is  based 
upon  the  arrangement  of  their  organs  of  motility,  as  follows: 

I.  Gymnobacteria,  possessing  no  flagella. 

II.  Trichobacteria,  with  flagella. 

1.  Monotricha,  having  a  single  flagellum  at  one  pole. 

2.  Lophotricha,  having  a  tuft  of  flagella  at  one  pole. 

3.  Amphitricha,  with  flagella  at  both  poles. 

4.  Peritricha,   with   flagella   completely   surrounding   the    bac- 

terial body. 
Bacterial  Spores. — ^A  large  number  of  bacteria  possesses  the  power  of 
developing  into  a  sort  of  encysted  or  resting  stage  by  a  process  commonly 
spoken  of  as  sporulation  or  spore  formation.  The  formation  of  spores 
by  bacteria  depends  largely  upon  environmental  conditions,  and  the 
optimum  environment  for  spore  formation  differs  greatly  for  varioua 
species.  It  is  usually  necessary  that  a  temperature  of  over  20°  C, 
exist  in  order  that  spores  may  be  formed.  Unfavorable  factors,  likcs^ 
acid  formation,  accumulation  of  bacterial  products  in  old  cultures,  or 

1  Me^sea,  Cent.  f.  Bakt.,  I,  Ref.  ix,  1891. 


16  BIOLOGY  AND  TECHNIQUE 

lack  of  nutrition,  frequently  seem  to  constituti^  the  stimuli  which  lead 
to  sporulation.  In  the  case  of  some  species,  notably  the  anthrax  bacillus, 
spores  are  formed  only  in  the  presence  of  free  oxygen  and  are  therefore 
never  formed  within  the  tissues  of  infected  animals.  It  is  claimed  that 
some  of  the  pathogenic  anaerobes,  like  B.  tetani  and  the  bacillus  of 
malignant  edema,  may  form  spores  anaerobically.  Nevertheless  it  has 
been  observed  that  when  an  absolute  exclusion  of  oxygen  is  practiced 
in  the  cultivation  of  these  bacteria,  vegetative  forms  only  are  seen  in 
the  cultures.^ 

The  process  of  sporulation  is  by  no  means  to  be  regarded  as 
a  method  of  multiplication,  since  it  rarely  occurs  that  a  single  bacil- 
lus  produces  more  than  one  spore.  In  some  species  of  bacteria  the 
formation  of  several  spores  in  one  individual  has  occasionally  been 
observed,  but  there  can  be  no  question  about  the  fact  that  such  a 
condition  is  exceptional. 

Varieties  of  spores  are  often  recognized,  the  so-called  arthrospores 
and  the  true  spores  or  endospores.  It  is  seriously  in  doubt  whether  the 
structures  once  spoken  of  as  arthrospores  should  be  considered  as  in  any 
way  comparable  to  true  spores.  They  are  represented  by  the  granular 
and  globular  appearances  occasionally  observed  in  old  cultures  of  some 
bacteria,  notably  streptococcus,  cholera  spirillum,  diphtheria  bacillus, 
and  others.  It  was  believed  that  they  were  due  to  a  transformation  of 
certain  individuals  of  the  cultures  into  more  resistant  forms.  It  ia 
probable,  however,  that  such  structures  are  merely  to  be  regarded  as 
evidences  of  involution  or  degeneration,  since  it  has  never  been  demon- 
strated that  cultures  containing  them  are  more  resistant  either  to  dis- 
infectants or  to  heat,  than  cultures  showing  no  evidences  of  such  forms. 
The  true  spores  or  endospores  are  most  common  among  bacilli,  and 
are  rarely  observed  among  the  spherical  bacteria.  They  arise  within 
the  body  of  the  individual  bacterium  as  a  small  granule  which  probably 
represents  a  concentration  of  the  protoplasmic  substance.  Nakanishi  ^ 
claims  that  there  is  a  definite  relation  between  these  sporogenic  globules 
and  the  nuclear  material  of  the  bacterial  cell.  At  the  time  at  which 
sporulation  occurs  there  is  usually  a  slight  and  gradual  thickening  of  the 
bacillary  body.  After  the  formation  of  this  thickening,  a  spore  mem- 
brane appears  about  the  same  thickened  area.  The  completed  spore  is 
usually  round  or  oval,  has  an  extremely  high  refractive  index,  and  a 


'^Zinsser,  Jour.  Exp.  Med.,  viii,  1906,  p.  542. 
» Nakanishi,  Munch,  med.  Woch.,  1900,  p.  680. 


MORPHOLOGY,  REPRODUCTION,  ETC, 


17 


D  0  H? 


Fig.  4.- 


Various  Positions  of  Spores 
IN  Bacterial  Cell. 


0000<?CP 


^l 


membrane  which  is  very  resistant.  '^MuhlschlegeP  believes  that  the  spore 
membrane  is  a  double  structure,  and,  as  stated  before,  Nakanishi  believes 
that  the  spore  contains  nuclear  material. 

The  position  of  the  spore  in  the  mother  cell  is  of  some  differential 
importance  in  that  it  is  usually  con- 
stant for  one  and  the  same  species. 
Thus,  the  spores  of  the  tetanus 
bacillus  are  regidarly  situated  at 
the  extreme  ends  of  the  bacillary 
bodies,  while  those  of  anthrax  are 
situated  at  or  near  the  middle. 

Physiologically,  sporulation  is  probably  to  be  regarded  as  a  method 
of  encystment  for  the  purpose  of  resisting  unfavorable  environment, 
^  and  it   is   indeed    true   that   species 

of  bacteria  the  vegetative  forais  of 
which  are  rather  easily  injured  by 
heat,  light,  drying,  and  chemicals 
have  a  comparatively  enormous  re- 
sistance to  these  agents  after  the 
formation  of  spores.  Thus,  while  a 
10-per-cent  solution  of  carbolic  acid 
will  kill  the  vegetative  forms  of 
anthrax  bacilli  within  twenty  min- 
utes, anthrax  spores  are  able  to  resist 
the  same  disinfectant  for  a  long 
period  in  a  concentration  of  over  50 
per ^ cent;  and  while  the  vegetative 
forms  of  the  same  bacillus  show  little 
more  resistance  against  moist  heat 
than  other  vegetative  forms,  the 
spores  will  withstand  the  action  of 
live  steam  for  as  long  as  ten  to  twelve 
minutes  and  more. 

Whenever  the  spores  of  any  mi- 
croorganism are  brought  into  an  en- 
vironment suitable  for  bacterial  growth  as  to  temperature,  moisture, 
and  nutrition,  the  spores  develop  into  vegetative  forms.  This  process 
differs  according  to  species.    In  general  it  consists  of  an  elongation  of 


hoi  A  ^/ 1 


B 


SO 


aO    lO 


Fig.  5. — Germination  op  Spores. 
A,  Bacillus  subtilis,  equatorial  spore 
germination;  B,  Bacillus  anthracis, 
germination  by  simple  transition;  G 
Clostrydium  butyricum,  polar  germi- 
nation. 


Muhischlegel,  Cent.  f.  Bakt.,  II  Abt.,  vi,  1900,  p.  65. 


18  BIOLOGY  ANP  TECHNIQUE 

the  spore  body  with  a  loss  of  its  highly  refractile  character  and  resist- 
ance to  staining  fluids.  The  developing  vegetative  cell  may  now 
rupture  and  slip  out  of  the  spore  membrane  at  one  of  its  poles,  leav- 
ing the  empty  spore  capsule  still  visible  and  attached  to  the  bacillary 
body.  Again,  a  similar  process  may  take  place  equatorially  instead 
of  at  the  pole.  In  other  species  again,  there  may  be  no  rupture  of 
the  spore  membrane  at  all,  the  vegetative  form  arising  by  gradual 
elongation  of  the  spore  and  an  absorption  or  solution  of  the  mem- 
brane which  is  indicated  by  change  in  staining  reaction.  Division  by 
fission  in  the  ordinary  way  then  ensues. 

REPRODUCTION  OF  BACTERIA 

Bacteria  multiply  by  cell  division  or  fission.  A  young  individual 
increases  in  size  up  to  the  limits  of  the  adult  form,  when,  by  simple 
cleavage,  at  right  angles  to  the  long  axis,  without  any  discoverable 
process  of  mitosis  or  nuclear  changes,  it  divides  into  two  individuals. 
In  spite  of  the  claims  of  various  bacteriologists,  notably  Nakanishi,^ 
traceable  analogy  to  the  karyokinesis  of  other  cells  has  not  been 
definitely  established.  In  the  case  of  the  spherical  bacteria  a  slight 
change  to  the  elliptical  .form  takes  place  just  before  cleavage  and 
this  cleavage  may  occur  in  one  plane  only,  in  two  planes,  or  in  three 
planes.  According  to  the  limitations  of  cleavage  direction,  the  cocci 
assume  a  chained  appearance  (streptococci),  a  grape-like  appearance 
(staphylococci),  or  an  arrangement  in  packets  or  cubes  having  three 
dimensions  (sarcinse).  In  the  cases  of  bacilli  and  spirilla,  cleavage 
takes  place  in  the  direction  of  the  short  axis.  The  individuals,  after 
cleavage,  may  separate  from  each  other,  or  may  remain  mutually 
coherent.  The  cohesion  after  cleavage  is  pronounced  in  some  species  of 
bacteria  and  slight  in  others,  and,  together  with  the  plane  of  cleavage, 
determines  the  morphology  of  the  cell-groups.  Thus  among  the  cocci 
diplo-  or  double  forms,  long  chains  and  short  chains  may  arise  and  fur- 
nish a  characteristic  of  great  aid  in  differentiation.  Similarly  among 
the  bacilli  there  are  forms  which  appear  characteristically  as  single 
individuals  and  others  which  form  chains  of  varying  length. 

The  rate  of  growth  varies  to  a  certain  extent  with  the  species,  and 
also  with  the  favorable  or  unfavorable  character  of  the  environment. 
A  generation,  that  is,  the  time  elapsing  in  the  interval  between  one 

*  Nakanishi,  Cent.  f.  Bakt.,  I,  xxx,  1901. 


MORPHOLOGY,  REPRODUCTION,  ETC.  1ft. 

cleavage  and  the  next,  has  been  estimated  by  A.  Fischer  ^  as  being 
about  twenty  minutes  for  the  cholera  spiriUum  and  16-20  minutes  for 
bacillus  coli  communis,  under  the  most  favorable  conditions.  The  same 
author  has  calculated  that  under  these  conditions  a  single  cholera 
spirillum  would  yield  1600  trillions  in  a  single  day.  Such  a  multiplica- 
tion rate,  however,  is  probably  not  usual  under  natural  or  even  artificial 
conditions,  both  on  account  of  lack  of  nutritive  material  and  because  of 
inhibition  of  the  growth  of  the  bacteria  by  their  own  products. 

VARIATIONS   OF   BACTERIAL   FORMS 

Variations  from  the  basic  forms  considered  in  the  preceding  sec- 
tion may  occur,  but  are  not  common  among  bacteria  under  normal 
conditions.    Thus  the  formation  of  club  shapes  by  a  thickening  of  the 


Fig.  6. — Degeneration  Forms  of  Bacillus  Diphtheri.e.    (After  Zettnow.) 

bacillary  body  at  one  or  both  ends  has  been  frequently  observed  among 
bacteria  of  the  diphtheria  group,  and  in  the  glanders  bacillus,  and  an 
irregular  beading  is  not  infrequently  observed  in  tubercle  bacilli  under 
normal  conditions.  Such  pictures  can  not,  in  these  cases,  be  regarded 
as  degeneration  or  involution  forms,  since  they  are  visible  in  young, 
actively  growing  cultures  under  ordinary  conditions.    It  is  a  well-known 

1  A.  Fischer,  "  Vorlesungen  uber  Bakt.,"  Jena,  1903. 


20 


BIOLOGY  AND  TECHNIQUE 


fact,  furthermore,  that  the  sizes  and  contours  of  bacteria  may  vary  to 
some  extent  according  to  the  medium  on  which  they  are  grown.  This 
may,  to  a  degree,  be  due  to  osmotic  relations.  On  fluid  media,  for  in- 
stance, many  bacteria  may  appear  larger  and  of  a  less  dense  consistency 
than  do  members  of  the  same  species  cultivated  upon  solid  media. 

Degeneration  Forms. — When  bacteria  are  grown  under  conditions 
which  are  not  entirely  favorable  for  their  development,  or  when  they 
are  grown  for  a  prolonged  period  upon  artificial  culture  media  without 
transplantation,  there  may  occur  variations  which  often  depart  consider- 
ably from  the  ground  type,  known  as  degeneration  or  involution  forms. 


Fig.  7. — Degeneration  Forms  of  Bacillus  Pestis.     (After  Zettnow.) 

Thus,  in  the  case  of  the  diphtheria  bacillus,  old  cultures  may  contain 
long,  irregularly  beaded  forms  with  broad  expansions  at  the  ends. 
In  the  case  of  B.  pestis  the  fact  that  large  numbers  of  oval,  vacuolated 
bodies  in  old  cultures  are  formed  regularly  has  become  of  differential 
value.^  These  degeneration  forms  are  shown  most  characteristically 
when  the  bacteria  are  cultivated  on  agar  containing  3  to  5  per  cent  NaCl. 
Among  the  cocci,  marked  evidences  of  involution  are  often  seen  in 
cultures  of  the  meningococcus  in  the  form  of  large,  swollen  poorly- 
staining  spheres,  and  in  the  case  of  the  pneumococcus  in  the  so-called 
shadow  forms  which  have  the  appearance  of  empty  capsules.   There  are 

*  Hankin  and  Leumanrif  Cent.  f.  Bakt.,  I,  xxii,  1897. 


MORPHOLOGY,  REPRODUCTION,  ETC.  21 

few  microorganisms  indeed,  in  which  prolonged  cultivation  on  artificial 
media  or  other  unfavorable  influences  do  not  produce  variations  from 
the  ground  type  which  may  often  make  the  cultures  morphologically 
unrecognizable.  In  the  case  of  many  of  the  spirilla  (spirillum  Milleri, 
spirillum  Metchnikovi,  etc.)  the  degeneration  forms  may  appear  within 
so  short  a  time  as  two  or  three  days  after  transplantation. 


CHEMICAL  AND  PHYSICAL  PROPERTIES  OF  THE  BACTERIAL  CELL 

Chemical  Constituents. — ^The  quantitative  chemical  composition  of 
bacteria  is  subject  to  wide  variations,  dependent  upon  the  nutritive 
materials  furnished  them. 

Approximately  80  to  85  per  cent  of  the  bacterial  body  is  water. 
The  remainder  consists  chiefly  of  proteids  which  constitute  roughly  from 
50  to  80  per  cent  of  the  dry  substances.  Remaining,  after  extrac- 
tion of  these,  are  fats,  and  in  some  cases  true  wax  (fatty  acid  combina- 
tions with  higher  alcohols),  traces  of  cellulose  (in  some  bacteria  only), 
and  the  ash  which  makes  up  usually  about  1  to  2  per  cent  of  the  dry 
substances. 

The  extensive  researches  of  Cramer^  have  shown  how  widely  at  va- 
riance quantitative  analyses  may  be  when  made  of  cultures  of  the  same 
species  of  bacteria  grown  upon  different  media.  Thus  the  dry  sub- 
stances of  the  cholera  vibrio  were  found  to  be  made  up  of  65  per  cent 
of  proteids  when  the  microorganisms  were  grown  upon  nutrient  broth 
as  against  45  per  cent  when  the  same  bacteria  had  been  grown  upon 
the  proteid-free  medium  of  Uschinsky.  Analyses  made  by  Kappes^  of 
B.  prodigiosus  and  by  Nencki  ^  and  Scheffer  of  some  of  the  putrefactive 
bacteria,  may  serve  to  illustrate  the  approximate  proportions  of  the 
substances  making  up  the  bacterial  body. 

B.  Putrefactive 

prodigiosus  Bacteria 

Water  85.45  per  cent.  83.42  per  cent. 

Proteids    10.33   "      "  13.96    "       " 

Fats 0.7     "      "  1.        "       " 

Ash   1.75   "      "  0.78   "       " 

Residue 1.77    "      "  0.84   "       " 


»  Cramer,  Arch.  f.  Hyg.,  xii,  xiii,  xvi,  xxii,  xxviii. 

^Kappes,  "  Analyse  der  Massenkulturen,"  etc.    Diss.,  Leipzig,  1889. 

8  Nencki  und  Scheffer,  Jour,  f .  prakt.  Chemie,  new  ser.  xix,  1880. 


22  BIOLOGY  AND  TECHNIQUE 

Analyses  of  the  tubercle  bacillus  by  Ruppel/  Hammerschlag,^  Weyl,^ 
and  others,  have  yielded  the  following  approximate  results  (calculated 
from  results  of  above-mentioned  authors). 

Tubercle  bacillus 

Water   85      to  86      per  cent. 

Proteids    8.5  to    9        "       " 

Fat  and  waxes 3 . 5  to    4        "       " 

Ash  and  carbohydrates    1 . 2  to    1.4    "       " 

The  proteids  which  are  contained  in  the  bacterial  dry  substances 
consist  partly  of  nucleoproteids,  globulins,  and  proteids  differing  ma- 
terially from  those  ordinarily  met  with.  Ruppel,  in  an  analysis  of 
the  tubercle  bacillus,  obtained  the  following  values,  for  100  grams  of 
dried  tubercle  bacilli: 

Nucleic  acid 8.5  grams. 

(Tuberculinic  acid) 

Nucleoprotamin 25 . 5  " 

Nucleoproteid 23  " 

Albuminoids 8.3  " 

(Keratin,  etc.) 

Fat  and  wax 26 . 5  " 

Ash 9.2  " 

A  true  globulin  has  been  isolated  from  bacteria  by  Hellmich,^  and 
true  proteids,  coagulable  by  heat,  have  been  demonstrated  by  Buchner,^ 
in  the  "  Presssaft "  or  juice  obtained  by  subjecting  bacteria  to  mechanical 
pressure.  In  this  connection,  too,  we  should  not  fail  to  consider  the 
thermolabile  toxic  substances  contained  in  many  bacteria,  the  endo- 
toxins, which  though  of  uncertain  chemical  nature,  are  probably  pro- 
teid  in  composition.® 

The  fats  which  are  demonstrable  both  by  microchemical  methods, 
staining  with  Sudan  III.,  Scharlach  R.,  Osmic  acid,  and  by  alcohol- 
ether  extraction,  consist  of  fatty  acids,  true  fats,  and,  in  the  case  of  the 
tubercle  bacillus  at  least,  of  waxy  substances.^ 


1  Ruppel,  Zeit,  f.  physiol.  Chemie,  xxvi,  1898. 
«  Hammerschlag ,  Zeit.  f,  klin.  Med.,  1891. 
»  Weyl,  Deut.  med.  Woch.,  1891. 

*  Hellmich,  Arch.  f.  exper.  Pathol.,  etc.,  xxvi. 

*  Buchner,  Munch,  med.  Woch.,  1897. 

*  Shattock,  Lancet,  May,  1898. 

» De  Schweinitz  and  Dorset,  Cent.  f.  Bakt.,  I,  xxii,  1897. 


MORPHOLOGY,  REPRODUCTION,  ETC.  23 

The  carbohydrates  isolated  from  various  bacteria  consist  chiefly  of 
small  quantities  of  cellulose  and  allied  bodies,  presumably  concerned  in 
the  formation  of  the  bacterial  cell  membrane.  The  demonstration  of 
these  substances  has  been  successful  only  in  isolated  cases  and  has  not 
found  universal  confirmation. 

Glycogen-like  substances  hasre  been  demonstrated,  according  to 
A.  Fischer,^  in  B.  subtilis  and  B.  coli.  These  bacteria  stained  a  reddish 
brown  color  when  stained  with  iodin,  and  after  treatment  with  weak 
acids  were  shown  to  contain  dextrose. 

The  bacterial  ash,  remaining  after  removal  of  other  substances,  con- 
gists  largely  of  phosphates  and  chlorides  of  potassium,  sodium,  cal- 
cium, and  magnesium. 

Osmotic  Properties  of  the  Bacterial  Cell. — Like  all  other  animal  and 
vegetable  cells,  the  bacterial  cell  forms  in  itself  a  small  osmotic  unit 
which  reacts  delicately  to  differences  of  pressure  existing  between  its 
own  protoplasm  and  the  surrounding  medium.  The  perfect  and  normal 
morphology  of  a  microorganism,  therefore,  can  exist  only  when  the 
osmotic  pressure  within  the  protoplasm  of  the  cell  is  isotonic  or  equal 
to  that  of  its  own  environment.  The  changes  produced  in  the  morpho- 
logical relations  of  a  cell  when  transferred  from  one  environment  into 
another  of  varying  osmotic  pressure,  depend  intimately  upon  the 
"  permeability "  of  the  cell  membrane  for  different  substances.  When 
such  a  membrane  is  permeable  for  water  and  not  for  substances  in  solu- 
tion, it  is  technically  spoken  of  as  "semi-permeable."  Now,  as  a  matter- 
of  fact,  the  bacterial  cell  membrane  is  easily  permeable  for  water,  but 
its  permeability  differs  greatly  in  various  species  of  bacteria  for  other 
substances.  Thus,  for  instance,  the  cholera  vibrio  shows  great  perme- 
ability for  common  salt  and  B.  fluorescens  liquefaciens  shows  a  lower 
permeability  for  potassium  nitrate  than  do  many  other  bacteria.^ 

When  a  microorganism  is  suddenly  removed  from  an  environment 
of  low  osmotic  pressure  into  one  showing  a  high  pressure,  say,  from  a 
dilute  to  a  concentrated  solution  of  NaCl,  an  abstraction  of  water  from 
the  cell  occurs,  with  a  consequent  shrinkage  of  the  protoplasm  away 
from  the  cell  membrane.  This  process  is  spoken  of  as  "plasmolysis." 
Cell  death  does  not  usually  occur  with  plasmolysis,  but  by  slow  diffusion 
of  the  salt  itself  into  the  protoplasm,  the  equilibrium  may  eventually 
be  restored  and  the  normal  morphology  of  the  cell  resumed.    In  all  cases 

'  A.  Fischer,  "  Vorlesungen  tiber  die  Bakt,,"  Jena,  1903. 
*  Gottschlich,  in  Fliigge,  "  Mikroorganismen,"  i,  p.  91. 


24  BIOLOGY  AND  TECHNIQUE 

the  speed  and  completeness  of  the  return  to  normal  depends  upon  the 
permeability  of  the  cell  membrane  for  the  dissolved  substances.  There 
is  no  evidence  to  support  the  view  that  the  internal  pressure  of  a  cell 
may  be  in  any  way  increased  by  an  inherent  power  of  the  protoplasm 
independently  of  the  laws  of  diffusion.  As  a  general  rule,  old  cultures 
are  more  susceptible  to  plasmolysis  than  are  young  and  vigorous  strains. 
Spores  and,  according  to  A.  Fischer,^  flagella  are  much  less  susceptible 
to  osmotic  changes  than  are  the  vegetative  bodies. 

When,  on  the  other  hand,  bacteria  are  suddenly  removed  from  a 
medium  possessing  a  high  osmotic  pressure  to  one  comparatively  low, 
say,  from  a  concentrated  salt  solution  to  distilled  water,  a  bursting  of  the 
cell  may  occur,  a  process  spoken  of  as  "  plasmoptysis."  Plasmoptysis 
leads  to  cell  death,  and  is  probably  the  cause  of  the  death  of  micro^ 
organisms  so  often  observed  in  distilled-water  emulsions  of  bacteria. 

Other  Physical  Properties  of  Bacteria. — ^The  refractive  index  of  the 
vegetative  bacterial  body  is  low,  in  contrast  to  the  highly  refractive 
character  of  the  spores  and  flagella.  According  to  Fischer,  the  ectoplasm 
or  cell  membrane  shows  a  higher  index  than  does  the  endoplasm. 

The  specific  gravity  of  various  microorganisms  has  been  investi- 
gated by  Bolton,^  Rubner,^  and  others.  Some  of  Rubner's  results  are 
the  following: 

Gelatin  fluidifiers  .  .  .• Sp.gr.  1 .0651 

Gas  formers "     "    1 .0465 

Cultures  from  potato "     "    1 .038 

M.  prodigiosus "     "    1 .  054 


^  A.  Fischer,  quoted  from  Gottschlich  in  Fliigge,  "  Mikroorganismen,"  I,  p.  91. 
2  BoUon,  Zeit.  f.  Hyg.,  i,  1886.  »  Rubner,  Arch.  f.  Hyg.,  xi,  1890. 


CHAPTER  III 


THE  RELATION  OF  BACTERIA  TO   ENVIRONMENT,  AND  THEIR 

CLASSIFICATION 

NUTRITION   OF    BACTERIA 

Like  all  protoplasmic  bodies,  bacteria  consist  of  carbon,  oxygen, 
hydrogen,  and  nitrogen,  to  which  are  added  inorganic  salts  and  varying 
quantities  of  phosphorus  and  sulphur.  In  order  that  bacteria  may 
develop  and  multiply,  therefore,  they  must  be  supplied  with  these  sub- 
stances in  proper  quantity  and  in  forms  suitable  for  assimilation.  To 
formulate  definite  laws  based  on  chemical  structure  as  to  the  compounds 
suitable,  and  those  unsuitable  for  use  by  the  bacteria,  is  obviously  im- 
possible owing  to  the  great  metabolic  variations  existing  within  the 
bacterial  kingdom,  and  notable  attempts  to  do  so,  such  as  those  by 
.Ijoew,*  have  not  successfully  withstood  critical  inquiry. 

Carbon. — ^The  carbon  necessary  for  bacterial  nourishment  or  ana- 
bolism  may  be  obtained  either  directly  from  proteids,  carbohydrates, 
and  fats,  or  from  the  simpler  derivatives  of  these  substances.  Thus,  the 
amido-acids,  leucin  and  tyrosin,  ketons,  and  organic  acids,  like  tartaric, 
citric,  and  acetic  acids,  glycerin,  and  even  some  of  the  alcohols,  may 
furnish  carbon  in  a  form  suitable  for  bacterial  assimilation.  A  limited 
number  of  bacterial  species,  furthermore,  notably  the  nitrobacteria  of 
Winogradsky,  are  capable  of  obtaining  their  required  carbon  from 
atmospheric  CO 3,  and  possibly  from  other  simple  carbon  compounds 
added  to  culture  media.^ 

Oxygen. — Oxygen  is  obtained,  by  the  large  majority  of  bacteria, 
directly  from  the  atmosphere  in  the  form  of  free  O2.  For  many  micro- 
organisms, moreover,  the  presence  of  free  oxygen  is  a  necessary  condi- 
tion for  growth.  These  are  spoken  of  as  the  "obligatory  aerobes." 
Among  the  pathogenic  bacteria  proper,  many,  like  the  gonococcus, 
bacillus  influenzae,  and  bacillus  pestis,  show  a  marked  preference  for  a 
well-oxygenated  environment.     Probably  there  is  no  pathogenic  micro- 

1  Loew,  Cent.  f.  Bakt.,  I,  xii,  1892. 

2  Muntz,  Compt.  rend,  de  Tacad.  des  sciences,  t.  iii, 

25 


26  BIOLOGY  AND  TECHNIQUE 

organism  which,  under  certain  conditions  of  nutrition,  is  entirely 
unable  to  exist  and  multiply  in  the  complete  absence  of  this  gas.  The 
conditions  existing  within  the  infected  animal  organism  cause  it  to 
seem  likely  that  all  incitants  of  infection  may,  at  times,  thrive  in  the 
complete  absence  of  free  oxygen. 

There  is  another  class  of  organisms,  on  the  other  hand,  for  whose 
development  the  presence  of  free  oxygen  is  directly  injurious.  These 
microorganisms,  known  as  "obligatory  anaerobes,"  obtain  their  supply 
of  oxygen  indirectly,  by  enzymatic  processes  of  fermentative  and  pro 
teolytic  cleavage,  from  carbohydrates  and  proteids,  or  by  reduction 
from  reducible  bodies.  Among  the  pathogenic  microorganisms  the  class 
of  "  obligatory  anaerobes  "  is  represented  chiefly  by  Bacillus  tetani,  the 
bacillus  of  malignant  edema,  the  bacillus  of  symptomatic  anthrax. 
Bacillus  aerogenes  capsulatus,  and  Bacillus  botulinus. 

Intermediate  between  these  two  classes  is  a  large  group  of  bacteria 
which  thrive  well  both  under  aerobic  and  anaerobic  conditions.  Some 
of  these,  which  have  a  preference  for  free  oxygen  but  nevertheless 
possess  the  power  of  thridng  under  anaerobic  conditions,  are  spoken 
of  as  "facultative  anaerobes."  In  others  the  reverse  of  this  is  true; 
these  are  spoken  of  as  "facultative  aerobes."  These  varieties  of 
bacteria  are  by  far  the  most  numerous  and  comprise  most  of  our 
parasitic  and  saprophytic  bacteria. 

The  relation  of  microorganisms  to  oxygen  is  extremely  subtle,  there- 
fore, and  not  to  be  biologically  dismissed  by  a  rigid  classification  into 
aerobes,  facultative  anaerobes,  and  obligatory  anaerobes.  Both  Engel- 
mann,*  by  a  method  of  observing  motile  bacteria  in  the  hanging  drop 
as  to  their  behavior  in  relation  to  the  oxygen  given  off  by  a  chlorophyll- 
bearing  alga,  and  Beijerinck,^  by  a  macroscopic  method  of  observing 
similar  bacteria  as  to  their  motion  away  from  or  toward  an  oxygenated 
area,  were  able  to  demonstrate  delicately  graded  variations  between 
species,  favoring  various  degrees  of  oxygen  pressure. 

The  discovery  by  Pasteur  that  certain  bacteria  develop  only  in  the 
absence  of  free  oxygen,  produced  a  revolution  in  our  conceptions  of 
metabolic  processes,  since  up  to  that  time  it  was  believed  that  life  could 
be  supported  only  when  a  free  supply  of  O2  was  obtainable.  Pasteur's 
original  explanation  for  this  phenomenon  was  that  anaerobic  conditions 
of  life  were  always  associated  with  some  form  of  carbohydrate  fermenta- 

1  Engelmann,  Botanische  Zeitung,  1881. 
a  Beijerinck,  Cento  f.  Bakt.,  I,  xiv,  1893. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  27 

tion  and  that  oxygen  was  obtained  by  these  microorganisms  by  a  split- 
ting of  carbohydrates.  As  a  matter  of  fact,  for  a  large  number  of  micro- 
organisms, this  is  actually  true,  and  the  presence  of  readily  fermentable 
carbohydrates  not  only  increases  the  growth  energy  of  a  large  number 
of  anaerobic  bacteria,  but  in  many  cases  permits  otherwise  purely 
aerobic  bacteria  to  thrive  under  anaerobic  conditions.^  On  the 
other  hand,  the  basis  of  anaerobic  growth  can  not  always  be  found 
in  the  fermentation  of  carbohydrates  or  in  the  simple  process  of 
reduction. 

The  favorable  influence  of  certain  actively  reducing  bodies,  like 
sodium  formate  or  sodium-indigo-sulphate,  upon  anaerobic  cultivation 
is  probably  referable  to  their  ability  to  remove  free  oxygen  from  the 
media  and  thus  perfect  the  anaerobiosis.^  A  number  of  strictly  anae- 
robic bacteria,  however,  may  develop  in  the  entire  absence  of  carbohy- 
drates or  reducing  substances,  obtaining  their  oxygen  supply  from  other 
suitable  sources,  some  of  which  may  be  the  complex  proteids.  Thus 
the  tetanus  bacillus  may  ^  thrive  when  the  nutritive  substances  in  the 
media  are  entirely  proteid  in  nature.      (See  p.  28.) 

As  Hesse  ^  has  shown,  the  respiratory  processes  of  aerobic  bacteria 
consist  in  the  taking  in  of  oxygen  and  the  excretion  of  CO2.  The  CO2 
excretion  has  been  shown,  in  these  cases,  to  be  markedly  less  than  is 
represented  in  the  intake  of  oxygen. 

Anaerobes,  likewise,  show  an  excretion  of  CO2  which  must,  in  these 
cases,  be  a  result  of  bacterial  katabolism. 

Certain  bacteria,  like  the  red  sulphur  bacteria,  have  the  power  of 
utilizing  atmospheric  oxygen  in  the  same  way  in  which  this  process 
takes  place  in  the  chlorophyll-bearing  plants. 

While  a  profuse  supply  of  oxygen  absolutely  inhibits  the  growth  of 
most  anaerobes,  a  number  of  these  may,  nevertheless,  develop  when  only 
small  quantities  of  oxygen  are  present.  Minute  quantities  of  free  oxy- 
gen in  culture  media  have  been  shown  by  Beijerinck^  and  others  not  to 
inhibit  the  growth  of  Bacillus  tetani  and  Theobald  Smith  •^  has  recently 
demonstrated  that  when  suitable  nutritive  material  in  the  form  of  fresh 
liver  tissue  is  added  to  bouillon,  a  number  of  anaerobic  bacteria  may  be 

• 
1  Theobald  Smith,  Cent.  f.  Bakt.,  I,  xviii,  1895. 
^Kitasato  and  Weyl,  Zeit.  f.  Hyg.,  viii,  1890. 
3  Chudiakow,  Cent.  f.  Bakt.,  Ref.,  II,  iv,  189a 
'^  Hesse,  Zeit.  f.  Hyg.,  xv,  1897. 
6  Beijerinck,  Cent.  f.  Bakt.,  II,  vi,   X900. 
"  Th,  Smith,  Brown,  and  Walker,  Jour.  Med.  Res.,  ix,  1906. 


28  BIOLOGY  AND  TECHNIQUE 

induced  to  grow  in  indifferently  anaerobic  environment.  Ferran,  * 
succeeded  in  adapting  the  tetanus  bacillus  to  an,  aerobic  environment. 
In  this  case,  however,  the  virulence  of  the  bacillus  was  lost. 

Nitrogen. — The  nitrogen  required  by  bacteria  is  taken,  in  most  cases, 
from  proteins,  and  many  of  the  non-diffusible  albumins  may  be  rendered 
assimilable  by  the  proteolyzing  enzymes  possessed  by  microorganisms. 
Among  the  pathogenic,  more  strictly  parasitic  bacteria,  moreover,  a 
delicate  specialization  may  be  observed  as  to  the  particular  varieties  of 
animal  albumin  which  may  be  utilized  by  them.  Thus  the  gonococcus 
grows  more  readily  only  upon  uncoagulated  human  blood  serum;  the 
Pfeiffer  bacillus  requires  hemoglobin,  and  the  diphtheria  bacillus  out- 
grows other  bacteria  upon  a  medium  composed  for  the  greater  part  of 
coagulated  beef  serum.  For  bacteria  that  do  not  require  native  animal 
protein  for  their  development,  the  most  common  nitrogenous  ingredient 
of  culture  media  is  pepton. 

Many  bacteria  (pathogenic  and  saprophytic),  on  the  other  hand, 
may  thrive  on  media  containing  absolutely  no  protein,  in  which  case, 
of  course,  a  synthesis  of  protein  must  be  assumed.  A  medium  used  to 
demonstrate  this,  devised  by  XJschinski,^  contains  ammonium  lactate, 
glycerin,  asparagin  (the  amide  of  amido-succinic  acid),  and  inorganic 
salts. 

Creatin,  creatinin,  urea  and  urates,  and  even  ammonia  compounds 
and  nitrates,  may  serve  as  sources  of  nitrogen  for  many  of  the  less 
parasitic  bacteria.  A  limited  number  of  species,  moreover,  the  bacilli 
in  the  root  tubercles  of  the  leguminosae  and  the  nitrogen-fixing  organ- 
isms of  the  soil,  can  obtain  their  nitrogen  directly  from  the  free  N2  of 
the  atmosphere. 

Although  the  sources  of  carbonaceous  and  of  nitrogenous  food  have 
been  separately  discussed,  it  should  not  be  forgotten  that,  in  many 
instances,  both  elements  are  taken  up  within  the  same  compound,  and 
that  separate  supplies  are  a  necessity  in  isolated  cases  only. 

Hydrogen. — Hydrogen  is  obtained  by  bacteria  largely  in  combina- 
tion as  water  and  together  with  the  carbon  and  nitrogen  containing 
substances. 

Salts. — The  phosphatic  constituents  of  the  bacterial  body  are  taken 
in,  chiefly,  as  phosphates  of  magnesium,  calcium,  sodium,  or  potassium. 
The  phosphates  seem  to  be  necessary  constituents  of  culture  media, 

1  F&rran,  Cent.  f.  Bakt.,  I,  xxiv,  1898. 

2  Uschinski,  Cent.  f.  Bakt.,  I,  xiv,  1893. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  29 

while  chlorides,  on  the  other  hand,  according  to  Proskauer  ^  and  Beck 
are  not  absolutely  essential.  Sodium  salts,  as  a  rule,  seem  to  be  more 
advantageous  for  purposes  of  bacterial  cultivation  than  potassium  salts. 

The  uncombined  sulphur,  which  is  often  a  constituent  of  bacteria, 
is  usually  supplied  by  soluble  sulphates.  In  the  case  of  the  thiobacteria 
of  Winogradsky,  however,  free  H2S  is  necessary  for  its  formation.^ 

The  iron  contained  in  some  higher  bacteria  is  taken  in  as  ferrous 
compounds,  and  is  oxidized  in  the  bacterial  into  ferric  compounds. 

The  relative  quantities  of  various  nutrients  in  culture  media  are 
important  in  so  far  as  too  high  concentrations  may  inhibit  growth.  In 
this  respect,  however,  separate  species  vary. 

The  development  of  bacteria  is  far  oftener  arrested  by  the  accumula- 
tion of  waste  products  than  by  an  exhaustion  of  nutrient  materials. 

PARASITISM  AND  SAPROPHYTISM 

When  we  speak  of  bacteria  as  parasites  or  as  saprophytes,  we  classify 
them,  primarily,  according  to  their  relationship  to  the  bodies  of  higher 
animals.  ''Parasites"  are  those  bacteria  which  are  capable  of  living 
and  multiplying  within  the  human  or  animal  body,  whereas  the  term 
"saprophytes"  refers  to  the  multitude  of  microorganisms  which  are 
unable  to  hold  their  own  under  the  environmental  conditions  found  in 
the  tissues  of  higher  animals,  but  are  found,  almost  ubiquitously,  in  air, 
soil,  manure,  and  water.  The  separation  is  by  no  means  a  sharp  one 
and  carries  with  it  other  implications,  which  the  use  of  these  terms  always 
conveys.  While  parasites  are  usually  very  fastidious  as  to  nutritional 
and  temperature  requirements,  most  saprophytes  are  easily  cultivated 
upon  the  simplest  media.  Thus  certain  parasitic  bacteria,  such  as  the 
bacillus  of  influenza,  the  gonococcus,  and  others,  are  dependent  upon 
specific  forms  of  animal  proteids  for  their  food  supply,  while  typical 
saprophytes,  like  Bacillus  proteus,  may  thrive  and  multiply  upon  even 
the  simplest  organic  proteid  derivatives. 

Between  the  strict  parasites  and  the  saprophytes,  there  is  a  large 
class  of  bacteria,  to  which  the  majority  of  pathogenic  varieties  belong,  the 
members  of  which  are  capable  of  developing  luxuriantly  under  both  con- 
ditions.    These  bacteria  are  often  spoken  of  as  facultative  parasites. 

More  recently  the  question  of  parasitism  and  saprophytism  has 

1  Proskauer  and  Beck,  Zeit.  f.  Hyg.,  xviii,  1895. 

2  Voges,  Cent.  f.  Bakt.,  I,  xviii,  1893. 


30  BIOLOGY  AND  TECHNIQUE 

become  closely  interwoven  with  our  conceptions  of  virulence.  Bail 
(see  section  on  Aggressins)  has  classified  parasites  into  strict  parasites 
and  half  parasites.  By  the  first  term  he  designates  bacteria  like  Bacillus 
anthracis,  which  actually  invade  all  the  tissues  of  their  host,  while,  by 
the  term  ''half  parasites,"  he  refers  to  microorganisms  like  the  spirilum 
of  cholera  which  gain  a  foothold  upon  some  part  of  the  body  of  the  host, 
but  do  not  actually  penetrate  into  the  general  circulation. 

All  pathogenic  bacteria,  therefore,  must  be  grouped  as  parasites, 
strict  or  facultative,  while  the  saprophytes,  as  a  class,  perform  the  far 
more  thankful  task  of  breaking  up  organic  matter  outside  of  the  animal 
body,  by  putrefaction  and  fermentation.  Absolute  separation  between 
the  two  classes,  however,  can  not  be  maintained,  since  many  ordinarily 
saprophytic  bacteria  may  display  parasitic  qualities  if  administered  in 
large  numbers  to  animals  or  man  in  whom  resistance  to  bacterial  invasion 
is  at  a  low  ebb. 


ANTAGONISM  AND  SYMBIOSIS  OF  BACTERIA 

The  ubiquity  of  bacteria  in  nature  of  course  implies  the  simul- 
taneous presence  of  many  species  in  all  places  where  special  conditions 
have  provided  a  favorable  environment  for  growth.  Thus  bacteriologi- 
cal investigation  of  water,  milk,  manure,  soil,  or  organic  infusions,  always 
reveals  the  presence  of  a  large  number  of  different  varieties  within  the 
same  substance.  If  the  food  supply  in  such  a  natural  culture  is  at  all 
limited  in  quantity,  or  the  removal  of  waste  products  is  prohibited,  it 
will  usually  be  found  that  gradually  the  numbers  of  varieties  will  dimin- 
ish and  a  few  species,  or  even  only  one,  will  prevail.  In  the  case  of  milk, 
for  instance,  after  standing  for  three  or  four  days  at  a  suitable  temper- 
ature, two  or  three  varieties  will  be  found  to  have  taken  the  place  of 
the  twenty  or  thirty,  which  may  have  been  present  originally. 

This  behavior  is  due  to  the  influence  which  various  microorganisms 
exert  upon  each  other  and  is  known  as  antagonism.  Such  antagonism 
probably  depends  upon  the  fact  that  the  metabolic  products  of  the  pre- 
dominant species  (the  one  or  ones  for  whom  the  special  cultural  condi- 
tions are  most  favorable)  inhibit  the  growth  of  the  less  vigorous  varieties. 
Many  examples,  experimentally  supported,  of  such  antagonism,  can  be 
given.  Thus,  the  gonococcus  is  distinctly  inhibited  by  the  soluble  pro- 
ducts of  Bacillus  pyocyaneus,  ^  while  in  the  presence. of  pyogenic  cocci  it 

1  Schafer,  Fortschr.  d.  Med.,  5,  1896. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  31 

develops  luxuriantly,  and  the  bacillus  of  plague  is  completely  inhibited 
when  streptococci  are  present  in  the  culture.  ^ 

Mutual  inhibition  may  also  be  due  to  the  monopoUzing  of  the  nutri- 
tion in  the  medium  by  the  predominating  species  or  to  the  change  in  re- 
action produced  by  its  growth.  This  last  consideration  is  probably  the 
secret  of  the  inhibitory  effect  exerted  by  acid-producers  upon  bacteria  of 
putrefaction,  and  has  received  practical  therapeutic  application  in 
Metchnikoff 's  lactic-acid  bacillus  therapy,  which  see. 

When  simultaneous  presence  of  two  species  in  the  same  environment 
favors  the  development  of  both,  the  condition  is  spoken  of  as  symbiosis. 
Such  dependence  is  not  so  frequent  as  antagonism,  but  it  does  occur. 
Examples  of  such  a  condition  have  been  observed  in  cultures  containing 
diphtheria  bacilH  and  streptococci  ^  and  have  been  frequently  observed 
in  cultures  containing  both  aerobic  and  anaerobic  bacteria,  where  the 
former  favor  the  development  of  the  latter  by  monopolizing  the  supply 
of  free  oxygen.  Symbiosis  may  also  take  place  in  cultures  in  which  com- 
plex food  products  are  split  up  by  one  species,  furnishing  substances  for 
ingestion  by  species  with  a  lesser  digestive  ability. 

RELATIONS   OP   BACTERIA  TO   PHYSICAL  ENVIRONMENT 

Relation  of  Temperature. — Like  all  other  living  beings,  bacteria 
develop  and  multiply  by  virtue  of  a  series  of  chemical  and  physical 
processes,  by  means  of  which  growth  energy  is  obtained  by  destruction 
or  catabolism,  and  the  lost  tissues  resupplied  by  absorption  of  nutritive 
materials.  It  is  natural,  therefore,  that  the  conditions  of  external 
temperature  should  intimately  affect  the  metabolic  processes.  The 
range  of  temperature  at  which  bacteria  may  grow  is  subject  to  wide 
variations  among  different  species.  Each  species,  on  the  other  hand, 
may  thrive  within  a  more  or  less  elastic  range  of  temperature,  each  one 
having  an  optimum,  a  minimum,  and  a  definite  maximum  tempera- 
ture. When  the  optimum  temperature  is  present  in  the  environment, 
the  functions  of  absorption  and  excretion  keep  pace  with  each  other,  and 
the  chemical  balance  is  well  preserved.  When  the  temperature  is  lower 
than  the  optimum,  all  metabolic  processes  take  place  more  slowly,  and 
the  bacterium  gradually  enters  into  a  resting  or  latent  stage,  at  which  ac- 
tual growth  may  be  exceedingly  slow  or  entirely  inhibited.  When  the 
temperature  is  higher  than  the  optimum,  the  destructive  processes  are 

1  Bitter,  Rep.  Egypt  Plague  Com.,  Cairo,  1897. 

2  Hubert,  Zeit.  f .  Hyg.,  xxix,  1895. 
4 


32  BIOLOGY  AND  TECHNIQUE 

carried  on  more  rapidly  than  the  substitution  of  waste  products  by  ab- 
sorption, and  a  gradual  weakening  of  vital  energy,  or  even  a  gradual 
death  of  the  bacterium,  may  take  place.  When  certain  bacteria  form 
spores,  they  become  very  much  more  resistant  against  both  high  and 
low  temperatures,  probably  because  a  true  resting  stage  has  been' 
reached,  during  which  metabolism  has  been  reduced  to  a  minimum, 
there  being  practically  no  nutritive  material  taken  in  and  corresponding- 
ly little  destruction  taking  place  within  the  body  of  the  microorganism. 

The  optimum  temperature  for  various  bacteria  depends  upon  the 
habitual  environment,  in  which  the  particular  species  is  accustomed  to 
exist.  Thus,  for  the  large  majority  of  bacteria  pathogenic  for  human 
beings,  the  optimum  temperature  is  at  or  about  37.5°  C.  There  are 
a  large  number  of  bacteria  common  in  water,  however,  which  grow 
hardly  at  all  at  the  body  temperature,  but  thrive  most  luxuriantly  at 
temperatures  of  about  20°  C.  F.  Forster,  ^  moreover,  described  certain 
phosphorescent  bacteria,  isolated  from  sea-water,  which  grow  readily  at 
0°  C,  or  a  little  above.  On  the  other  hand,  Miquel  ^  has  described  non- 
motile  bacilli,  which  he  isolated  from  the  water  of  the  Seine,  which  grew 
rapidly  at  temperatures  ranging  about  70°  C,  and  the  so-called  "muce- 
dinees  thermophiles, ''  described  by  Tsiklinski,^  develop  most  readily  at 
temperatures  very  little  above  this.  It  is  thus  plain  that  the  tempera- 
tures favored  by  various  bacteria  depend  to  a  large  extent  upon  an 
adaption  of  these  bacteria  through  many  generations  to  specific  en- 
vironmental conditions.  A  good  illustration  of  this  is  furnished  by  the 
bacillus  of  avian  tuberculosis,  a  microorganism  differing  essentially 
from  the  bacillus  of  himaan  tuberculosis  in  that  its  optimum  growth 
temperature  lies  at  41°^2°  C,  a  temperature  which  exceeds  the  op- 
timum temperature  for  the  human  type  by  as  much  as  the  normal  tem- 
perature of  birds  exceeds  that  of  man.  The  same  principle  is  illustrated 
by  the  facts  that  the  bacteria  which  have  a  very  low  optimum  tem- 
perature are  usually  those  isolated  from  water,  and  the  so-called  ther- 
mophile  or  high-temperature  bacteria  are  obtained  from  hot  springs  and 
from  the  upper  layers  of  the  soil,  where,  according  to  Globig,*  occasion- 
ally temperatures  ranging  from  about  55°  C.  occur. 

As  stated  before,  one  and  the  same  species  may  develop  within  a 


1  F.  Forster,  Cent,  f .  Bakt.,  ii,  1887. 

2  Miquel,  Bull,  de  la  Stat.  Munic.  de  Paris,  1879. 
'  Tsiklinski,  Ann.  Past.,  1889. 

«(?fo%,  Zeit.  f.  Hyg.,  iii. 


RELATION  TO  ENVmONMENT— CLASSIFICATION  33 

wide  temperature  range,  and  it  may  be  possible,  by  persistent  cultiva- 
tion at  special  temperatures,  to  adapt  certain  bacteria  to  grow  luxu- 
riantly at  temperatures  removed  by  several  degrees  from  their  normal 
optimum.  In  such  cases  it  may  often  occur  that  special  characteristics 
of  the  given  species  may  be  lost.  An  example  of  this  is  the  loss  of  viru- 
lence and  of  spore-formation  which  takes  place  when  anthrax  bacilli 
are  cultivated  at  42°  C,  or  the  loss  of  the  power  to  produce  pigment 
when  bacillus  prodigiosus  is  grown  at  temperatures  above  30°  C. 

The  vegetative  forms  of  most  of  the  pathogenic  bacteria  may  grow 
at  temperatures  ranging  between  20°  C.  and  40°  C.  This  can,  however, 
by  no  means  be  regarded  as  applicable  to  all  of  the  pathogenic  bacteria, 
as  some  of  these,  like  the  gonococcus,  the  pneumococcus,  the  tubercle 
bacillus,  and  others,  are  delicately  susceptible  to  temperature  changes 
and  have  the  power  of  growing  only  within  limits  varying  but  a  few 
degrees  from  their  optimum.  Others,  on  the  other  hand,  like  bacilli  of 
the  colon  group,  Bacillus  anthracis.  Spirillum  cholerae  asiaticse,  etc., 
may  develop  at  temperatures  as  low  as  10°  C.  and  as  high  as  40°  C,  or 
over.  The  range  of  temperature  at  which  saprophytic  bacteria  may 
develop  is  usually  a  far  wider  one.  When  temperatures  exceed  in  any 
considerable  degree  the  maximum  growth  temperature,  the  vegetative 
forms  of  bacteria  perish.  Thus,  ten  minutes'  exposure  to  a  temperature 
of  between  55°  and  60°  C.  causes  death  of  the  vegetative  forms  of  most 
microorganisms.  Death  in  such  cases  is  due  probably  to  a  coagulation 
of  the  protoplasm,  and  since  all  such  processes  of  coagulation  take  place 
best  in  the  presence  of  water,  the  thermal  death  point  of  most  bacteria 
is  lower  when  heat  is  applied  in  the  form  of  boiling  water  or  steam, 
than  when  employed  as  dry  heat.  ,  (See  section  on  Sterilization.) 

When  spores  are  present  in  cultures,  the  resistance  to  heat  is  enor- 
mously increased.  Exactly  what  the  explanation  of  this  is  can  not  be  at 
present  stated.  It  may  be  that  the  high  concentration  in  which  the 
protoplasmic  mass  is  found  in  the  spores  renders  it  less  easily  coagulable 
than  is  the  protoplasm  of  the  vegetative  body.  A  more  detailed  discus- 
sion of  these  relations  will  be  found  in  the  section  on  Heat  sterilization. 

The  thermal  death  points  of  many  bacteria  have  been  carefully 
studied  by  Sternberg,^  by  a  technique  described  elsewhere. 

The  thermal  death  points  ascertained  by  him  in  this  way,  with  an 
exposure  of  ten  minutes  in  a  fluid  medium,  for  some  of  the  more  common 
non-sporogenic  bacteria  are  as  follows : 


Sternberg,  "Textbook  of  Bacteriology,"  New  York,  1901. 


H  BIOLOGY  AND  TECHNIQUE 

Spirillum  cholerae  asiaticae 52°  C 

Diplococcus  pneumoniae 52°  C. 

Streptococcus  pyogenes 54°  C. 

Bacillus  typhosus 56°  C. 

Bacillus  pyocyaneus 56°  C. 

Bacillus  mucosus  capsulatus 56°  C. 

Bacillus  prodigiosus 58°  C. 

Staphylococcus  pyogenes  aureus 58°  C. 

Gonococcus 60°  C. 

Staphylococcus  pyogenes  albus 62°  C. 

The  bacillus  tuberculosis,  though  not  a  spore  bearer,  seems  to  be  slightly 
more  resistant  to  heat  than  other  purely  vegetative  microorganisms. 
Thus,  according  to  Smith  ^  and  others,  ten  and  twenty  minutes'  ex- 
posure to  a  temperature  of  70°  C.  is  necessary  to  destroy  tubercle  bacilli 
in  a  fluid  medium.  For  the  effectual  destruction  of  spores  by  moist 
heat,  a  temperature  of  100°  C,  or  boiling  point,  is  usually  necessary. 

Low  temperatures  are  much  less  destructive  than  the  high  ones,  and 
are  even  in  a  number  of  cases  useful  in  keeping  bacteria  alive  for  long 
periods,  inasmuch  as  metabolic  processes  are  inhibited  and  life  is  main- 
tained without  actual  development  in  a  sort  of  resting  state.  Actual 
destruction  by  low  temperatures  rarely  takes  place.  The  exposure  of 
diphtheria,  tj^hoid,  and  other  bacilli  to  temperatures  as  low  as  200°  C. 
below  zero  has  been  carried  out  without  destruction  of  the  microorgan- 
isms, a  fact  which  is  of  great  importance  in  consider'ng  the  possibility 
of  infection  by  the  vehicle  of  ice.  Meningococci  and  gonococci,  on  the 
other  hand,  die  out  rapidly  when  exposed  to  0°  C. 

Relation  to  Pressure. — High  pressure  does  not  injure  bacteria. 
Certes  ^  found  that  a  pressure  of  two  atmospheres  had  no  influence 
upon  the  growth  of  anthrax  bacilli  suspended  in  blood. 

Relation  to  Moisture.— For  the  growth  and  development  of  all  bac- 
teria, the  presence  of  water  is  necessary.  Nutritive  materials  can  not 
be  absorbed  by  an  osmotic  process  unless  in  a  state  of  solution.  While 
complete  dryness  does  not  permit  growth,  its  destructive  action  upon 
various  bacteria  is  subject  to  great  differences.  The  effect  of  complete 
drying  upon  bacteria  will  be  found  more  fully  discussed  on  page  62.  In 
the  same  place  may  be  found  a  discussion  of  the  effects  of  light,  elec- 
tricity, x-ray,  and  radium  rays  upon  bacteria. 


1  Th.  Smith,  Jour,  of  Experimental  Med.,  No.  3,  1899. 

2  Certes,  Compt.  rend,  de  I'acad.  d.  sc,  99,  Paris,  1884. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  35 

THE    CLASSIFICATION   OF   BACTERIA 

Too  simple  in  structure,  too  varied  in  biological  properties  to  be 
definitely  identified  with  either  the  vegetable  or  animal  kingdom,  the 
bacteria  are  placed  at  the  bottom  of  the  scale  of  all  living  beings.  Closely 
linked  on  the  one  hand  to  the  plant  kingdom  by  the  yeasts  and  the 
molds,  and  on  the  other  to  the  animal  kingdom  by  the  protozoa,  they 
themselves  combine,  within  one  and  the  same  division,  attributes  so 
widely  divergent  as  to  structure,  metabolism,  and  biological  activity  that 
their  grouping  is  more  a  matter  of  working  convenience  than  of  actual 
scientific  classification.  Thus,  for  instance,  all  stages  of  metabolic  ac- 
tivity fill  in  the  gap  between  the  synthetizing  sulphur  and  nitrifying 
bacteria  and  the  purely  katabolic  activities  of  some  of  the  aerobic  and 
anaerobic  microorganisms  which  cause  putrefaction.  Growth  takes 
place  within  the  limits  of  a  wide  temperature  range,  and  the  specific 
modes  of  life  and  cultural  conditions  are  subject  to  the  widest  varia- 
tions, from  those  of  an  indisputably  useful  saprophytism  to  those  of  the 
most  exquisite  parasitism.  Although,  therefore,  strictly  speaking,  the 
bacteria  can  be  classified  as  a  whole  neither  in  the  animal  nor  in  the 
vegetable  realms,  being  nonchlorophyll-bearing,  they  are  for  conve- 
nience classified  with  the  fungi  or  colorless  plants. 

The  relationship  of  the  bacteria  to  other  simple  plants  may  be 
graphically  represented  by  the  following  scheme: 

Cryptogamia. 

I 
Thallophyta. 

I 


Alg^.  Lichens.  Fungi. 

! 

SCHIZOMYCETES  BlASTOMYCETES  HyPHOMYCETES 

(Bacteria).  (Yeasts).  (Moulds — Oidia). 

Coccacea.  Chlamydobacteria. 

Bacteriaceae.  (Higher  bacteria.) 

Spirillacese.  Streptothrix. 

Cladothrix. 
Leptothrix. 
Actinomyces. 

The  special  classification  of  the  bacteria  has  offered  still  greater 
difficulties,  for  the  lower  we  proceed  in  the  phylogenetic  scale  of  living 
beings,  the  less  specialized  the  morphological  and  biological  charac- 
teristics of  any  group  become,  and  the  more  difficult  it  is  to  establish  a 


36  BIOLOGY  AND  TECHNIQUE 

classification  which  can  in  any  way  be  regarded  as  final.  It  is,  there- 
fore, quite  impossible  to  classify  the  bacterial  varieties  or  species  on  any 
basis  which  can  hope  to  satisfy  all  the  demands  of  scientific  accuracy 
and  it  is  necessary  to  resort  to  the  expedient  of  utilizing  some  one 
characteristic  which  remains  constant  for  the  individual  genus  and  to 
base  upon  this  an  attempt  at  grouping.  When  bacteria  were  first  dis- 
covered, and  for  many  years  following,  numerous  observers  contended 
that  the  form  of  the  microorganism  observed  was  not  a  constant  one 
for  each  genus,  but  that  cocci  could  be  converted  into  bacilli  or  spirilla 
according  to  environmental  conditions.  It  was  Cohn^  who,  in  1872, 
first  recognized  the  constancy  of  the  morphology  of  bacteria  and  es- 
tablished, upon  morphological  basis,  a  classification  which,  with  minor 
changes,  has  been  retained  until  the  present  day..  Such  classifications 
can  not,  however,  be  regarded  as  anything  more  than  a  convenient 
make-shift  pending  the  day  when  the  finer  structure  and  true  biological 
relations  of  the  various  bacteria  shall  have  been  more  accurately  inves- 
tigated. The  scheme  most  commonly  accepted  at  present  is  the  one 
given  below,  proposed  by  Migula  2: 

Bacteria  (Schizomycetes).— Fission  fungi  (chlorophyll  free),  cell  division  in  one, 
two,  or  three  directions  of  space.  Many  varieties  possess  power  of  form- 
ing endospores.  Whenever  motility  is  present,  it  is  due  to  flagella,  or, 
more  rarely,  to  undulating  membranes. 

Family  I.  Coccace^. — Cells  in  free  state  spherical.    Division  in  one,  two, 

or  three  directions  of  space,  by  which  each  cell  divides  into  two,  four,  or 

eight  segments,  each  of  which  again  develops  into  a  sphere.    Endospore 

formation  rare. 
Genits  I.  Streptococcus. — Cells  divide  in  one  direction  of  space  only,  for 

which  reason,  if  they  remain  connected  after  fission,  bead-like  chains  may 

be  formed.    No  organs  of  locomotion. 
Genus  II.  Micrococcus  (Staphylococcus). — Cells  divide  in  two  directions 

of  space,  whereby,  after  fission,  tetrad  and  grape-like  clusters  may  be 

formed.    No  organs  of  locomotion. 
Genus  III.  Sardna. — Cells  divide  in  three  directions  of  space,  whereby, 

after  fission,  bale-like  packets  are  formed.    No  organs  of  locomotion. 
Genus  IV.  Planococcus. — Cells  divide  in  two  directions  of  space,  as  in 

micrococcus,  but  possess  flageUa. 


^K!ohn,  "Beitrage  zur  Biol.  d.  Pflanzen,"  Heft  1  u.  2,  1872. 
^Migvla,  "System  d.  Bakt.,"  Jena,  1897. 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  37 

Geniis  V.  Planosarcina. — Cells  divide  in  three  directions  of  space  as  in 
sarcina,  but  possess  flagella. 

Family  II.  BACTERiACEiE.— Cells  long  or  short,  cyhndrical,  straight,  never 
spiral.  Division  in  one  direction  of  space  only,  after  preliminary  elonga- 
tion of  the  rods. 

Geniis  I.  Bacterium. — Cells  without  flagella,  often  with  endospores. 

Genus  II.  Bacillus. — Cells  with  peritrichal  flagella,.  of  ten  with  endospores. 

Genus  III.  Pseudomonas. — Cells  with  polar  flagella.  Endospores  occur 
in  a  few  species,  but  are  rare. 

Family  III.  Spirillace.b. — CeUs  spirally  curved  or  representing  a  part  of  a 
spiral  curve.  Division  in  one  direction  of  space  only,  after  preceding 
elongation  of  cell. 

Genus  I.  Spirosoma. — Cells  without  organs  of  locomotion.    Rigid. 

Genus  II.  Microspira. — Cells  rigid,  with  one  or,  more  rarely,  two  or  three 
polar  undulated  flageha. 

Genu^  III.  Spirillum. — Cells  rigid,  with  polar  tufts  of  five  to  twenty 

.    flagella  usually  curved  in  semicircular  or  flatly  undulating  curves. 

Genus  IV.  Spirochcete. — Cells  sinously  flexible.  Organs  of  locomotion  un- 
known, perhaps  a  marginal  undulating  membrane. 

t*AMiLY  IV.  CHLAMYDOBACTERiACEiE. — ^Forms  of  Varying  stages  of  evolution, 
all  possessing  a  rigid  sheath  (Hiille),  which  surrounds  the  cells.  Cells 
united  in  branched  or  unbranched  threads. 

Genus  I.  Streptothrix. — Cells  united  in  simple,  unbranched  threads.  Divi- 
,  sion  in  one  direction  of  space  only.  Reproduction  by  non-motile 
conidia. 

Genus  II.  Cladothrix. — Cells  united  or  pseudodichotomously  branching 
threads.  Division  in  one  direction  of  space  only.  Vegetative  multipli- 
cation by  separation  of  entire  branches.  Reproduction  by  swarming 
forms  with  polar  flagella. 

Genus  III.  Crenothrix. — Cells  united  in  unbranched  threads,  at  first  with 
division  in  one  direction  of  space  only.  Later  the  cells  divide  in  all  three 
directions  of  space.  The  daughter  cells  become  rounded  and  develop 
into  reproductive  cells. 

Genus  IV.  Phragmidiothrix. — Cells  at  first  united  in  unbranched  threads, 
dividing  in  three  directions  of  space,  thus  forming  a  rope  of  cells.  Later 
some  of  the  cells  may  penetrate  through  sheath,  and  thug  give  rise  to 
branches. 


38  -       .       BIOLOGY  AND  TECHNIQUE 

Genits  V.  Thiothrix. — Unbranched,  non-motile  threads,  inclosed  in  fine 
sheaths.  Division  of  cells  in  one  direction  only.  Cells  contain  sulphur 
granules. 

Family  V.  Beggiatoace^e. — Cells  united  in  sheathless  threads.     Division 
in  one  direction  of  space  only.     Motihty  by  undulating  membrane  as  in 
Oscillaria. 
Genus  Beggiatoa. — Cells  with  sulphur  granules. 

It  will  be  seen  in  reviewing  the  classification  just  given  that  the  sub- 
divisions are  based  upon  questions  of  form,  motility,  and  situation  of 
flagella.  While  these  characteristics,  so  far  as  we  know,  are  constant, 
there  are,  nevertheless,  many  instances  in  which  types  entirely  similar 
in  these  respects  must  be  differentiated.  This  can  be  done  only  by  care- 
ful study  of  staining  reactions,  finer  structure,  cultural  characteristics, 
and  biological  activities. 

As  a  matter  of  fact,  while  the  botanical  classification  of  the  bacteria 
offers  great  difficulties,  identification  is  not  so  complicated  a  task  as  this 
would  indicate.  Identification,  once  roughly  made  on  a  morphological 
basis,  is  further  carried  on  by  the  aid  of  cultural  characteristics,  by  bio- 
chemical reactions  and  by  pathogenic  properties.  The  bacteria  occupy 
so  important  a  place  in  agriculture,  in  medicine,  and  in  hygiene,  that  it 
rarely  becomes  necessary  for  a  worker  in  any  particular  field  to  survey 
the  entire  group.  The  habitat  of  a  large  number  of  species  is  so  well 
known  that  this  consideration  alone  often  gives  a  clew  to  actual  identi- 
fication. 

Bacterial  Mutation. — The  earlier  views  of  bacteriologists  concerning 
mutation  differed  greatly,  Naegeli  holding  that  extensive  mutation  was 
probably  the  rule;  Cohn,  on  the  other  hand,  holding  strictly  to  the 
constancy  of  form  and  species.  The  accumulated  experience  of  many 
bacteriologists  during  the  years  since  then  seems  to  point  almost  entirely 
in  the  direction  indicated  by  Cohn,  and,  in  fact,  most  of  our  methods  of 
classification  are  based  upon  the  assimiption  of  such  constancy. 

Form  alone,  of  course,  cannot  be  relied  upon  for  classification  among 
organisms  so  simply  constructed  that  the  possibilities  of  variation  in 
form  are  very  limited.  In  classifying  bacteria,  therefore,  we  are  forced 
to  take  cognizance  not  only  of  morphology,  but  also  of  staining  character- 
istics, behavior  on  differential  media,  fermentation  reactions,  patho- 
genicity, •  and,  as  a  final  appeal,  reactions  with  specific  immune  sera. 
The  last  especially,  as  utilized  in  agglutination  and  complement-fixation, 


RELATION  TO  ENVIRONMENT— CLASSIFICATION  39 

seems  to  indicate  a  fundamental  chemical  difference  in  the  constitution 
of  bacteria  often  morphologically  very  much  alike.  It  is  certainly  a 
remarkable  fact  that  organisms  such  as  those  belonging  to  the  colon- 
typhoid-dysentery  group,  though  morphologically  not  differentiable,  may 
still  retain  differences  both  in  pathogenicity  and  in  fermentation  powers 
after  being  kept  for  ten  or  more  years  in  laboratory  media,  and  the  same 
experience  we  have  had  with  organisms  belonging  to  the  diphtheria 
group.  The  virulence  of  plague  and  anthrax  bacilli  may  be  retained 
for  years  in  storage,  and  such  evidence  shows  pretty  definitely  that 
fundamental  constant  differences  between  organisms  exist. 

In  judging  of  mutation  we  must  differentiate  between  temporary 
changes  of  secondary  characteristics  which  revert  to  the  type  rapidly 
when  brought  back  to  the  normal  environment  and  those  which  consti- 
tute permanent  inherited  characteristics.  Of  recent  years  much  work 
has  been  done  on  this  question,  which  has  been  reviewed  very  thoroughly 
by  Eisenberg  ^  and  by  Vaughan.  ^  Systematic  cultivation  of  colon  and 
typhoid  bacilli  in  the  hands  of  Twort,  Penfold  and  others  seems  to  have 
shown  that  agglutination  as  well  as  fermentation  characteristics  can  be 
artificially  changed.  Furthermore,  color-producing  organisms  Uke  the 
prodigiosus  can  be  artificially  changed  to  colorless  strains,  and  it  is 
well  known  that  certain  microorganisms  rapidly  lose  their  virulence 
when  cultivated,  and  that  the  virulence  can  only  be  brought  back  by 
passage  through  animals.  Rosenow^  claims  recently  to  have  converted 
hemolytic  streptococci  into  typical  streptococcus  viridans,  pneumococ- 
cus  mucosus,  and  pneumococcus-like  organisms.  In  just  how  far  these 
observations  will  be  shown  to  represent  true  permanent  mutations  we 
are  not  at  present  ready  to  determine.  If  it  will  be  found  that  organisms 
typically  representative  of  a  well-known  species  can  be  changed  in  the 
animal  body  or  in  culture  into  forms  recognizedly  typical  of  another 
species,  we  will  have  to  revise  our  classifications,  and  we  can  look  upon 
the  classes  as  now  established  merely  as  convenient  methods  of  making 
discussion  possible,  but  not  as  representing  botanically  constant  types. 

While  we  must  therefore  admit  that  a  considerable  degree  of  muta- 
tion is  possible,  we  do  not  ourselves  believe  that  the  evidence  is  suffi- 
ciently strong  to  undermine  the  prevailing  ideas  as  to  the  constancy  of 
species.  Most  mutations  so  far  produced  have  readily  reverted  to 
type  when  subjected  to  proper  conditions. 

1  Eisenberg,  Weichhardt's  Ergebnisse,  1914. 

2  Vaughan,  Jour,  of  Lab.  &  Clin.  Med.,  1915,  voL  i,  145. 
'  Rosmow,  Jour.  Infect.  Dis.,  xiv,^1914,  1. 


CHAPTER  IV 

THE  BIOLOGICAL  ACTIVITIES   OF  BACTERIA 

While  the  bacteria  pathogenic  to  man  and  animals  largely  usurp 
the  attention  of  those  interested  in  disease  processes,  this  group  of  micro- 
organisms is  after  all  but  a  small  specialized  off-shoot  of  the  realm  of 
bacteria,  and,  broadly  speaking,  actually  of  minor  importance.  Sur- 
veying the  existing  scheme  of  nature,  as  a  whole,  it  is  not  an  extrava- 
gant statement  to  say  that  without  the  bacterial  processes  which  are 
constantly  active  in  the  reduction  of  complex  organic  substances  to 
their  simple  compounds,  the  chem^jal  interchange  between  the  animal 
and  vegetable  kingdoms  would  fail,  and  all  life  on  earth  would  of 
necessity  cease.  To  understand  the  full  significance  of  this,  it  is  neces- 
sary to  consider  for  a  moment  the  method  of  the  interchange  of  matter 
between  the  animal  and  vegetable  kingdoms. 

All  animals  require  for  their  sustenance  organic  compounds.  They 
are  unable  to  build  up  the  complex  protoplasmic  substances  which  form 
their  body  cells  from  chemical  elements  or  from  the  simple  inorganic 
salts.  They  are  dependent  for  the  manufacture  of  their  food-stuffs, 
therefore,  directly  or  indirectly,  upon  the  synthetic  or  anabolic  activi- 
ties of  the  green  plants. 

These  plants,  by  virtue  of  the  chlorophyll  contained  within  the  cells 
of  their  leaves  and  stems,  and  under  the  influence  of  sunlight,  possess 
the  power  of  utilizing  the  carbon  of  the  carbonic  acid  gas  of  the  atmos- 
phere, and  of  combining  it  with  water  and  the  nitrogenous  salts  ab- 
sorbed by  their  roots,  building  up  from  these  simple  radicles  the  highly 
complex  substances  required  for  animal  sustenance. 

These  products  of  the  synthetic  activity  of  the  green  plants,  then, 
are  ingested  by  members  of  the  animal  kingdom,  either  directly,  in  the 
form  of  vegetable  food,  or  indirectly,  as  animal  matter.  They  are 
utilized  in  the  complex  laboratory  of  the  animal  body  and  are  again 
broken  down  into  simpler  compounds,  which  leave  the  body  as  excreta 
and  secreta. 

The  excreta  and  secreta  of  animals,  however,  are,  in  a  small  part 
only,  made  up  of  substances  simple  enough  to  be  directly  utilized  by 
plants.    The  dead  bodies,  moreover,  of  both  animals  and  plants  would 

40 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  41 

be  of  little  further  value  as  stores  of  matter  unless  new  factors  inter- 
vened to  reduce  them  to  that  simple  form  in  which  they  may  again 
enter  into  the  synthetic  laboratory  of  the  green  plant.  Agents  for 
further  cleavage  of  these  compounds  are  required,  and  these  are  supplied 
by  the  varied  activities  of  the  bacteria. 

On  the  other  hand,  bacteria  are  also  important  in  the  process  of 
synthesis.  The  main  supply  of  nitrogen  available  for  plant  life  is  found 
in  the  elementary  state  in  the  atmosphere — a  condition  in  which  it 
can  not  be  utilized  as  a  raw  product  by  the  plant.  This  gap  again  is 
bridged  by  the  bacteria  found  in  the  root  bulbs  of  the  leguminous  plants 
— bacteria  which  possess  the  power  of  assimilating  or  aiding  in  the  as- 
similation of  atmospheric  nitrogen  and  its  preparation  for  further  use  by 
the  plant  itself.  Another  bacterial  activity  which  may  be  classified  as  an 
anabolic  process  is  the  oxidation  of  the  ammonia,  released  by  decomposi- 
tion, into  nitrites  and  nitrates.  This  is  carried  on  by  certain  bacteria  of 
the  soil.    These  are  to  be  treated  of  in  greater  detail  in  another  section. 

There  is  a  constant  circulation,  therefore,  of  nitrogen  and  carbon 
compounds,  between  the  plant  and  the  animal  kingdoms,  by  virtue  of  an 
anabolic  or  constructive  process  in  the  one,  and  a  katabolic  or  destruc- 
tive process  in  the  other,  rendering  them  mutually  interdependent  and 
indispensable.  The  circuit,  however,  is  not  by  any  means  a  closed  one; 
there  are  important  gaps,  both  in  the  process  of  cleavage  and  in  that  of 
synthesis,  which,  if  left  unbridged  by  the  bacteria,  would  effectually 
arrest  all  life-activity  of  plants  and  eventually  of  animals. 

Far  from  being  scourges,  therefore,  these  minute  microorganisms 
are  paramount  factors  in  the.  great  cycle  of  living  matter,  supplying 
necessary  links  in  the  circulation  of  both  nitrogenous  and  carbon  com- 
pounds. 

KATABOLIC   ACTIVITIES   OF   BACTERIA 

The  katabolic  activities  of  bacteria,  then,  consist  in  the  fermentation 
of  carbohydrates  and  in  the  cleavage  of  proteids  and  fats. 

Fermentation  is  carried  out  to  a  large  extent 'by  the  yeasts,  but  also 
to  no  inconsiderable  degree  by  bacteria.  Proteid  decomposition  and  the 
cleavage  of  fats  are  carried  out  almost  exclusively  by  bacteria. 

For  our  knowledge  of  the  fundamental  laws  underlying  these  phe- 
nomena of  fermentation  and  proteid  decomposition,  we  are  indebted 
to  the  genius  of  Pasteur,^  who  was  the  first  to  prove  experimentally  the 

'  Pasteur,  "  fitude  sur  la  bi3re,"  Paris,  1876. 


4:2  BIOLOGY  AND  TECHNIQUE 

exclusive  and  specific  parts  played  by  various  microorganisms  in  these 
processes.  While  the  observations  and  deductions  made  by  Pasteur  have 
not  been  greatly  modified,  a  large  store  of  information  has  been  gained 
since  his  time,  which  has  thrown  additional  light  upon  the  chemical  de- 
tails and  the  more  exact  manner  of  action  of  the  factors  involved. 

The  actual  work  of  cleavage  in  both  fermentation  and  proteid  cleav- 
age is  carried  out  by  substances  known  as  enzymes  or  ferments,  the  nature 
of  which  we  must  further  discuss  before  their  manner  of  action  can  be 
fully  comprehended. 

Bacterial  Enzymes  or  Ferments. — ^A  ferment  or  enzyme  is  a  substance 
produced  by  a  living  cell,  which  brings  about  a  chemical  reaction  with- 
out entering  into  the  reaction  itself.  The  enzyme  itself  is  not  bound  to 
any  of  the  end  products  and  is  not  appreciably  diminished  in  quantity 
after  the. reaction  is  over,  although  its  activity  may  be  finally  inhibited 
by  one  or  another  of  the  new  products.  The  action  of  bacterial  enzymes  is 
thus  seen  to  be  closely  similar  to  that  of  the  chemical  agents  technically 
spoken  of  as  "katalyzers,''  represented  chiefly  by  dilute  acids.  Thus, 
if  an  aqueous  solution  of  saccharose  is  brought  into  contact  with  a 
dilute  solution  of  sulphuric  acid,  the  disaccharid  is  hydrolyzed  and  is 
decomposed  into  levulose  and  dextrose. 

Thus: 

In  contact  with  Dextrose         Levulose 

dilute  H2SO4 

During  this  process,  which  is  known  as  "inversion,"  the  concentration 
of  the  sulphuric  acid  remains  entirely  unchanged.  While  theoretically 
the  changes  brought  about  by  enzymes  and  katalyzers  are  usually 
such  as  would  occur  spontaneously,  the  time  for  the  spontaneous  oc- 
currence would  be,  at  ordinary  temperatures,  infinitely  long.  The  defini- 
tion for  enzymes  and  katalyzers  is  given  by  Ostwald,  therefore,  as 
*' substances  which  hasten  a  chemical  reaction  without  themselves  taking 
part  in  it."  Exactly  the  same  result  which  is  obtained  by  the  use  of  dilute 
sulphuric  acid  is  caused  by  the  ferment  "invertase"  produced,  for 
instance,  by  B.  megatherium.  Were  a  solution  of  saccharose  sub- 
jected to  heat,  without  katalyzer  or  ferment,  a  similar  change  would 
occur,  but  by  the  mediation  of  these  substances  the  inversion  is  pro- 
duced without  other  chemical  or  physical  reinforcement. 

This  analogy  between  enzymes  and  katalyzing  agents  is  very 
striking.     Thus,  as  stated,  both  katalyzers  and  enzymes  bring  about 


THE     BIOLOGICAL     ACTIVITIES     OF     BACTERIA  43 

changes  without  themselves  being  used  up  in  the  process,  both  act 
without  the  aid  of  heat,  and  the  reactions  brought  about  by  both 
have  occasionally  been  shown  to  be  reversible.  While  this  last  phe- 
nomenon has  been  variously  shown  for  katalyzers,  the  process  of  re- 
versibility has  been  demonstrated  for  bacterial  enzyme  action  only  in 
isolated  cases.  Thus,  it  has  been  found  that  by  the  action  of  the  yeast 
enzyme  maltase  upon  concentrated  dextrose  solutions,  a  re-formation  of 
maltose  may  occur.  In  both  cases,  moreover,  the  quantity  of  enzyme 
or  katalyzer  is  infinitely  small  in  proportion  to  the  amount  of  material 
converted  by  their  action. 

There  is  a  close  similarity,  furthermore,  between  the  bacterial  en- 
zymes and  the  ferments  produced  by  speciaHzed  cells  of  the  higher  ani- 
mals and  plants.  For  instance,  the  action  of  the  ptyalin  of  the  saliva  or 
of  the  diastase  obtained  from  plants  is  entirely  analogous  to  the  starch- 
splitting  action  of  the  amylase  produced  by  many  bacteria. 

The  action  of  all  enzymes  depends  most  intimately  upon  environ- 
mental conditions.  For  all  of  them  the  presence  of  moisture  is  essential. 
All  of  them  depend  for  the  development  of  their  activity  upon  the  exist- 
ence of  a  specifically  suitable  reaction.  Strong  acids  or  alkalies  always 
inhibit,  often  destroy  them.  Temperatures  of  over  70°  C.  permanently 
destroy  most  enzymes,  whereas  freezing,  while  temporarily  inhibiting 
their  action,  causes  no  permanent  injury,  so  that  upon  thawing,  their 
activity  may  be  found  almost  unimpaired.  Direct  sunlight  may  injure, 
but  rarely  destroys,  ferments.  Against  the  weaker  disinfectants  in  com- 
mon use,  enzymes  often  show  a  higher  resistance  than  do  the  bacteria 
which  give  rise  to  them. 

The  optimum  conditions  for  enzyme  action,  then,  consist  in  the 
presence  of  moisture,  the  existence  ot  a  favorable  reaction,  weakly  acid 
or  alkaline,  as  the  case  may  be,  and  a  temperature  ranging  from  35°- 
45°  C} 

Proteolytic  Enzymes. — In  nature,  the  decomposition  of  dead  animal 
and  vegetable  matter  occurs  only  when  the  conditions  are  favorable  for 
bacterial  development.  Thus,  as  is  well  known,  freezing,  sterilizing  by 
heat,  or  the  addition  of  disinfectants  will  prevent  the  rotting  of  organic 
material. 

In  the  laboratory,  the  presence  of  proteolytic  enzymes  is  determined 
chiefly  by  the  power  of  bacteria  to  Uquefy  gelatin,  fibrin,  or  coagulated 
blood  serum.    These  ferments  are  not  always  secretions  from  the  bac- 

» Oppenheimer,  "  Die  Fermente,"  etc.    Leipzig,  1900. 


44  BIOLOGY  AND  TECHNIQUE 

teria]  cell,  but  in  some  cases  may  be  closely  bound  to  the  ceIl4)ody  and 
separable  only  by  extraction  after  death.  In  such  cases  they  are  ^x>ken 
of  as  endoenzymes-  Whenever  they  are  true  secretory  products,  however, 
they  can  be  obtained  separate  from  the  microorganisms  which  form  them 
by  filtration  through  a  Berkef eld  candle.  From  such  fihrates  they  may, 
in  some  cases,  be  obtained  in  the  dry  state  by  precipitation  with  alcohoL 
When  obtained  in  this  way  the  precipitated  enzyme  is  usually  much 
more  thermostable  than  when  in  solution,  for  idiile  soluble  enzymes  in 
filtrates  are  usually  destroyed  by  70**  C,  and  even  less,  the  dried  powder 
may  occasionally  withstand  140**  C  for  as  long  as  ten  minutes.^ 

Apart  from  the  general  conditions  of  temperature  and  moisture,  the 
development  of  these  enzymes  seems  to  depend  directly  upon  the  presence 
of  proteids  in  the  culture  media.  The  number  of  bacterial  species 
which  produce  proteoiytic  enzymes  is  legion.  Among  those  more  com- 
mordy  met  with  are  staphylococci,  B.  subtilis,  B.  proteus,  B.  faecalis 
Hquefaciens,  Spirillum  cholerse  asiaticae,  B.  anthracis,  B.  tetani,  B.  pyo- 
cyaneus,  and  a  large  number  of  others.  The  inability  of  any  given  micro- 
organism to  Hquefy  gelatin  or  fibrin  by  no  means  entirely  excludes  the 
formation  by  it  of  proteolytic  enzymes,  since  these  ferments  may  often 
be  active  for  one  particular  class  of  proteid  only. 

In  order  to  study  the  qualitative  and  quantitative  powers  of  any 
given  bacterial  proteolyzing  enzjine  or  protease,  it  b,  of  course,  neces- 
sary to  study  these  processes  in  pure  culture  in  the  test  tube  with  media 
of  known  composition.  In  the  refuse  heap,  in  sewage,  or  in  rotting 
excreta,  the  process  is  an  extremely  compUcated  one,  for  besides  the 
bacteria  which  attack  the  proteid  molecule  itself,  there  are  many  other 
species  supplementing  these  and  each  other,  one  species  attacking  the 
more  or  less  complex  «[id-products  left  by  the  action  of  the  others. 

Exactly  what  the  chemical  reactions  are  which  take  place  in  these 
cleavages  is  not  entirely  clear.  It  is  believed,  however,  that  most  of  the 
cleavages  are  of  an  hydrohtic  nature. 

In  general,  the  action  of  the  proteid-splitting  f ermoits  is  ecMnpanUe 
to  that  of  the  pancreatic  ferment  trypsin,  and  they  are  most  aii&a.  active 
in  an  alkaline  enviroimientw  They  differ,  among  themselves,  chiefly  in 
the  form  of  proteid  which  they  are  competent  to  attack,  and  in  the 
extent  to  which  they  are  able  to  reduce  it  toward  its  simple  radicles. 

A  distinction  is  occasionally  made  between  the  terms  piartfaction 
and  decay,  the  former  being  used  to  rrfer  to  the  decompositiafi  taking 


THE    BIOLOGICAL    ACTIVITIES    OP    BACTERIA  45 

place  under  anaerobic  conditions,  that  is,  in  the  absence  of  oxygen,  a 
process  usually  resulting  in  incomplete  cleavage  of  the  proteid  medium; 
the  latter  being  used  to  signify  decompositions  under  aerobic  conditions 
and  leading  to  a  more  complete  splitting,  the  end-products  often  being 
represented  by  such  simple  compounds  as  carbon  dioxide,  water,  and 
ammonia.  In  general,  the  products  of  putrefaction  are  largely  repre- 
sented by  the  amino-acids,  leucin  and  tyrosin,  fatty  acids,  mercaptan, 
indol,  and  skatol.  The  gases  generated  in  such  decomposition  are  largely 
made  up  of  COj,  hydrogen,  NH^  and  HjS.  The  coincident  presence, 
furthermore,  of  the  carbohydrate-splitting  bacteria  and  of  denitrifying 
microorganisms  renders  the  actual  process  of  putrefaction  a  chaos  of 
many  activities  in  which  the  end-products  and  by-products  are  qualita- 
tively determinable  onlj^  with  much  inexactitude,  and  which  com- 
pletely defies  any  attempt  at  quantitative  analysis. 

Ptomains. — ^There  are  certain  products,  however,  resulting  from  the 
proteolytic  action  of  bacterial  enzjines  upon  proteids  which  claim  more 
than  a  purely  chemical  interest  because  of  their  toxic  action  upon  the 
animal  organism,  and  their  consequent  importance  as  incitants  of  dis- 
ease. Pre-eminent  among  these  are  the  ptomains.  The  word  ptomain 
(from  rrdi/xa,  a  dead  body)  is  used  to  designate  organic  chemical 
compounds  produced  by  the  action  of  bacteria,  which  are  basic  in  char- 
acter; that  is,  are  able  to  combine  with  an  acid  to  form  a  salt.  They 
should  be  definitely  distinguished  from  the  so-called  leucomains,  a 
term  employed  to  designate  similar  substances  formed  in  the  course 
of  proteid  metabolism  within  the  animal  body,  and  not  bacterial  in 
origin.  Both  in  their  basic  characters  and  in  their  nitrogenous  constitu- 
tion, the  ptomains  resemble  the  vegetable  alkaloids,  and  for  this  reason 
are  sometimes  spoken  of  as  "  animal  alkaloids." 

The  ptomains  must  be  sharply  distinguished  from  the  bacterial 
loxins,  which  are  products  of  the  bacterial  growth  irrespective  of  the 
medium  in  which  they  are  grown,  except  in  so  far  as  this  hinders  or 
abets  the  development  of  the  microorganisms.  Thus,  toxins  may  be 
developed  by  diphtheria  organisms,  for  instance,  in  proteid-free  media. 
As  will  be  seen  in  a  subsequent  section,  the  true  toxins  are  comparable 
to  the  enzymes  themselves,  rather  than  to  their  cleavage  products,  rep- 
resented in  this  instance  by  the  ptomains. 

A  great  number  of  ptomains  are  chemically  known.  Many  5)f 
these  possess  little  or  no  toxicity.  Others,  however,  like  putrescin 
(tetramethylenediamin,  C4H12N3)  and  cadaverin  (CsH.^Na)  are  very 
highly  poisonous.    It  is  to  one  or  another  of  these  ptomains  that  most 


46  BIOLOGY  AND  TECHNIQUE 

cases  of  so-called  meat  poisoning  (kreatoxismus) ,  cheese  poisoning 
(tyrotoxismus) ,  or  vegetable  poisoning  (sitotoxismus)  are  due. 

In  each  individual  case  the  variety  of  ptomain  resulting  from  a  bac- 
terial decomposition  varies  with  the  individual  species  of  microorganism 
taking  part  in  the  process  and  with  the  nature  of  the  proteid  upon  which 
its  development  takes  place. 

In  breaking  down  animal  excreta,  the  task  of  the  bacteria  is  rather 
a  simpler  one  than  when  dealing  with  the  cadavers  themselves,  for  here  a 
part  of  the  cleavage  has  already  been  carried  out  either  by  the  destruc- 
tive processes  accompanying  metabolism,  or  by  partial  decomposition  by 
bacteria  begun  within  the  digestive  tract.  This  material  outside  of  the 
body  is  further  reduced  by  bacterial  enzymes  into  still  simpler  sub- 
stances, the  nitrogen  usually  being  liberated  in  the  form  of  ammonia. 
One  example  of  such  an  ammoniacal  fermentation  may  be  found  in 
the  case  of  the  urea  fermentation  by  Micrococcus  urese,  in  which  the 
cleavage  of  the  urea  takes  place  by  hydrolysis  according  to  the  follow- 
ing formula: 

(NH2)2  CO  +  2H2  O  =  CO2  +  2NH3  +  H2  O 

Similar  ammoniacal  fermentations  are  carried  out,  though  perhaps 
according  to  less  simple  formulae,  by  a  large  number  of  microorganisms. 
Perhaps  the  most  common  species  which  possesses  the  power  is  the  group 
represented  by  B.  proteus  vulgaris  (Hauser). 

From  what  has  been  said  it  follows  naturally  that,  so  far,  the  decom- 
position of  the  proteid  molecule  from  its  complex  structure  to  ammonia 
or  simple  ammonia  compounds  is  an  indispensably  important  function, 
not  only  for  agriculture,  but  for  the  maintenance  of  all  life  processes. 
It  is  clear,  on  the  other  hand,  that  a  further  decomposition  of  ammonia 
compounds  into  forms  too  simple  to  be  utilized  by  the  green  plants  would 
be  a  decidedly  harmful  activity.  And  yet  this  is  brought  about  by  the 
so-called  denitrifying  bacteria  which  will  be  considered  in  a  subsequent 
section. 

Lab  Enzymes. — ^There  are  a  number  of  ferments  produced  by  bacteria 
which,  although  affecting  proteids,  can  not  properly  be  classified  with 
the  proteolytic  enzymes.  These  are  the  so-called  coagulases  or  lab 
enzymes,  which  have  the  power  of  producing  coagulation  in  liquid  pro- 
teids. Just  what  the  chemical  process  underlying  this  coagulation  is, 
is  not  known.     If  Hammarsten's  ^  conclusions  as  to  the  hydrolytic 

I  Hammarsten,  "Textbook  of  Physiol.  Chemistry,"  Translation  by  Mandel. 


THE     BIOLOGICAL     ACTIVITIES     OF     BACTERIA  47 

nature  of  the  changes  produced  by  them  are  true,  these  enzymes  are 
brought  into  close  relationship  to  the  proteolyzers,  although  a  coagula- 
tion can  hardly  be  regarded  as  a  true  katabolic  process.  In  milk  where 
the  lab-action  becomes  evident  by  precipitation  of  casein,  a  strict  dif- 
ferentiation must  be  made  between  this  coagulation  and  that  brought 
about  by  acids  or  alkalies.  In  the  former  case,  casein  is  not  only  pre- 
cipitated and  converted  into  paracasein,  but  is  actually  changed  so  that 
when  redissolved  it  is  no  longer  precipitated  by  lab.' 

Coagulating  enzymes  for  milk  proteids,  blood,  and  other  proteid 
solutions  are  produced  by  a  large  variety  of  bacteria.  They  have  been 
observed  in  cultures  of  the  cholera  vibrio,  B.  prodigiosus,  B.  pyocyaneus, 
and  several  others.^ 

The  lab  enzymes  are  easily  destroyed  by  temperatures  of  70°  C.  and 
over,  and  are  very  susceptible  to  excessive  acidity  or  alkalinity. 

Fat-Splitting  Enzymes  (Lipase). — The  fat-splitting  powers  of  bac- 
teria have  been  less  studied  than  some  of  the  other  bacterial  func- 
tions and  are  correspondingly  more  obscure.  It  is  known,  nevertheless, 
that  the  process  is  due  to  an  enzyme  and  that  it  is  probably  hydrolytic 
in  nature.  The  folio  wing,  formula  represents  the  simplest  method  in 
which  some  of  the  molds  and  bacteria  produce  cleavage  of  fats  into 
glycerin  and  fatty  acid. 

C3  H5  (C^  H,„_,  02)3  +  3H2  O  ==  C3  H3  (OH3)  +  3C^  H^n  O, 

Glycerin         Fatty  acid 

Some  of  the  bacteria  endowed  with  the  power  of  producing  lipase 
are  the  spirillum  of  cholera,  B.  fluorescens  liquefaciens,  B.  prodigiosus, 
B.  pyocyaneus.  Staphylococcus  pyogenes  aureus,  and  some  members  of 
the  streptothrix  family.  The  methods  of  investigating  this  function  of 
bacteria,  originated  by  Ejkmann, '  consists  in  covering  the  bottom  of  a 
Petri  dish  with  tallow  and  pouring  over  this  a  thin  layer  of  agar.  Upor 
this,  the  bacteria  are  planted.  Any  diffusion  of  lipase  from  the  bacterial 
colonies  becomes  evident  by  a  formation  of  white,  opaque  spots  in  the 
tallow.  Carriere  '*  was  able  to  demonstrate  a  fat-splitting  ferment  for  the 
tubercle  bacillus.  Apart  from  the  importance  of  these  enzymes  in 
nature  for  the  destruction  of  fats,  they  are  industrially  important  be- 

»  Oppenheimer,  "  Die  Fermente  u.  ihre  Wirkung,"  Leipzig,  1903. 
«  Tarini,  Atti  dei  laborat.  d.  sanita,  Rome,  1890. 
»  Ejkmann,  Cent.  f.  Bakt.,  I,  xxix,  1901. 
*  Carriere,  Comptes  rend,  de  la  soc.  de  biol.,  53, 1901. 
5 


48  BIOLOGY  AND  TECHNIQUE 

cause  of  their  action  in  rendering  butter,  milk,  tallow,  and  allied  prod- 
ucts rancid,  and  are  medically  of  interest  for  their  action  upon  fats  in 
the  intestinal  canal. 

Enzymes  of  Fermentation  {The  Cleavage  of  Carbohydrates  by  Bacterid). 
— The  power  to  assimilate  carbon  dioxide  from  the  atmosphere  is 
possessed  only  by  the  green  plants  and  some  of  the  colored  algae, 
and  the  sulphur  or  Thiobacteria.  All  other  living  beings  are  thus 
dependent  for  their  supply  of  carbon  upon  the  synthetic  activities 
carried  on  by  these  plants  to  the  same  degree  in  which  they  are  de- 
pendent upon  similar  processes  for  their  nitrogen  supply.  The  return  of 
this  carbon  to  the  atmosphere  is,  of  course,  brought  about  to  a  large  ex- 
tent by  the  respiratory  processes  of  the  higher  animals.  The  carbon, 
which,  together  with  nitrogen,  forms  a  part  of  proteid  combinations,  is 
freed,  as  we  have  seen  in  a  previous  section,  by  the  processes  of  proteid 
cleavage.  That,  however,  which  is  inclosed  in  the  carbohydrate  mole- 
cule, is  set  free  by  the  action  of  yeasts,  molds,  or  bacteria,  by  an  enzy- 
matic process  similar  in  every  respect  to  that  described  above  for  the 
process  of  proteid  cleavage. 

Fermentation. — The  power  of  carbohydrate  cleavage  is  possessed 
by  a  large  number  of  the  yeasts  and  bacteria.  The  process,  as  has 
been  indicated,  is  of  great  importance  in  the  cycle  of  carbon  compounds 
for  the  return  of  carbon  to  its  simplest  forms,  and  is,  furthermore,  as 
will  be  seen  in  a  later  section,  of  great  utility  in  the  industries.  In  each 
case  the  power  to  split  a  particular  carbohydrate  is  a  more  or  less  specific 
characteristic  of  a  given  species  of  microorganism,  and  for  this  reason 
has  been  extensively  used  as  a  method  for  the  biological  differen- 
tiation of  bacteria.  In  the  course  of  much  careful  work  upon  this 
question  it  has  been  ascertained  that  the  specific  carbohydrate-splitting 
powers  of  any  given  species  are  constant  and  unchanged  through 
many  generations  of  artificial  cultivation.  Thus,  differentiation  of  the 
Gram-negative  bacteria,  the  members  of  the  pneumococcus-streptococ- 
cus  group,  and  the  diphtheria  group,  can  now  largely  be  made  by  a  study 
of  their  sugar  fermentations. 

In  most  of  these  cases,  as  far  as  we  know,  the  cleavage  is  produced  by 
a  process  of  hydrolysis.  A  convenient  nomenclature  which  has  been 
adopted  for  the  designation  of  these  ferments  is  that  which  employs  the 
name  of  the  converted  carbohydrate  adding  the  suffix  "  ase  "  to  indicate 
the  enzyme.  There  are  thus  ferments  known  as  amylase,  cellulase,  lac- 
tase, etc. 

Amylase    (Diastase  or  Amylolytic  Ferment). — Amylases  or  starch- 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  49 

splitting  enzymes  are  formed  by  many  plants  (malt)  and  by  animal 
organs  (pancreas,  saliva,  liver).  Among  microorganisms  amylase  is 
produced  by  many  of  the  strep tothrix  group,  by  the  spirilla  of.  Asiatic 
cholera  and  of  Finkler-Prior,  by  B.  anthracis,  and  many  other  bacteria. 
A  large  number  of  the  bacteria  found  in  the  soil,  furthermore,  have 
been  shown  to  produce  amylases.  By  cultivating  bacteria  upon  starch- 
agar  plates,  amylase  can  be  readily  demonstrated  by  a  clearing  of  the 
medium  immediately  surrounding  the  colonies.^ 

Since,  of  course,  there  are  several  varieties  of  starches,  it  follows  that 
the  exact  chemical  action  of  amylase  differs  in  individual  cases.  The 
determination  of  the  structural  disintegration  of  starch  by  these  fer- 
ments is  fraught  with  much  difficulty,  owing  to  the  polymeric  constitu- 
tion of  the  starches.  Primarily,  however,  a  cleavage  takes  place  into 
a  disaccharid  such  as  maltose  (hexobiose) ,  and  the  non-reducing  sugars 
and  dextrin.  Beyond  this  point,  however,  the  further  cleavages  are 
subject  to  much  variation  and  are  not  entirely  clear.  The  dextrins 
upon  further  reduction  yield  eventually  dextrose. 

Cellulase. — Cellulose  is  fermented  by  a  limited  number  of  bacteria, 
most  of  them  anaerobes.  The  chemical  process  by  which  this  takes  place 
is  but  poorly  understood.^ 

Gelase. — An  agar-splitting  ferment  has  been  found  by  Gran.^ 

Invertase. — The  enzymes  which  hydrolytically  cause  cleavage  of 
saccharose  into  dextrose  and  levulose  are  numerous.  The  chemical 
process  takes  place  according  to  the  following  formula: 

Ci2  H22  On  +  H2  0  =  Cg  Hi2  Og  +  Cg  Hi2  Og 
Saccharose  Dextrose       Levulooe 

Invertase  is  produced  by  many  of  the  yeasts.  It  is  one  of  the  most 
common  of  the  enzymes  produced  by  bacteria,  and  has  been  found  in 
cultures  of  B.  megatherium,  B.  subtilis,  pneumococcus,  some  strepto- 
cocci, B.  coli,  and  many  others.  Invertase  is  usually  very  susceptible  to 
heat,  being  destroyed  by  temperatures  of  70°  C.  and  over.  A  slightly 
acid  reaction  of  media  abets  the  inverting  action  of  these  enzymes. 
Strong  acids  and  alkalies  inhibit  them.  Inverting  enzymes  may  be 
precipitated  out  of  solution  by  alcohol.  Antiseptics  even  in  weak  con- 
centrations will  inhibit  their  action. 


1  Ejkmann,  Cent.  f.  Bakt.,  xxix,  1901,  and  xxxv,  1904. 

2  Omelianski,  Lafar's  ''  Handb.  d.  techn.  Mykologie,"  Bd.  iii,  Chap.  9. 
»  Gran,  Bergens  Museum  Aarbog,  1902,  Hft.  I. 


50  BIOLOGY  AND  TECHNIQUE 

Lactase. — Lactose-splitting  ferments  are  extremely  common  both 
among  bacteria  and  among  the  yeasts.  The  process-  is  here  again  a 
hydrolytic  cleavage  resulting  in  the  formation  of  the  monosaccharids 
as  dextrose  and  galactose. 

Maltase. — ^A  maltose-splitting  ferment  has  also  been  found  in  the 
cultures  of  many  bacteria,  leading  to  the  formation  of  dextrose. 

Lactic  Acid  Fermentation. — Lactic  acid  (oxyproprionic  acid,  CgHg  O3) 
is  one  of  the  most  common  substances  to  appear  among  the  prod- 
ucts of  bacterial  activity,  both  in  media  containing  carbohydrates 
and  in  those  consisting  entirely  of  albuminous  substances.  In  most  of 
these  cases,  the  lactic  acid  is  formed  merely  as  a  by-product  accom- 
panying many  other  more  complicated  chemical  cleavages.  In  some 
instances,  however,  lactic  acid  is  produced  from  carbohydrates,  both 
disaccharids  and  monosaccharids,  as  an  almost  pure  product  due  to  a 
specific  bio-chemical  process.  The  reactions  taking  place  in  this  phenom- 
enon may  be  briefly  expressed  according  to  the  following  formulae: 

C,,R,,0,,  +  R,0=    4C3He03 
Lactose  Lactic  acid 

or 

Cq  H12  Og  =  2C3  Hg  O3 
Dextrose     Lactic  acid 

In  the  same  way  lactic  acid  may  be  produced  by  bacteria  from  levu- 
lose. 

Examples  of  lactic  acid  formation  are  furnished  by  the  streptococcus 
lacticus,  and  B.  lactis  aerogenes.  In  the  case  of  the  former,  the  fer- 
mentation may  indeed  proceed  by  the  simple  chemical  process  indi- 
cated in  the  formulae,  since  the  action  of  the  bacillus  is  entirely  unac- 
companied by  the  evolution  of  gas. 

Numerous  other  bacteria  produce  large  amounts  of  lactic  acid  from 
lactose,  possibly  by  chemical  processes  less  simply  formulated.  Among 
these  are  bacilli  of  the  colon  group,  B.  prodigiosus,  B.  proteus  vulgaris, 
and  many  others.  Although  lactic  acid  is  usually  the  chief  product  in 
the  bacterial  fermentation  of  the  simpler  carbohydrates,  acetic,  formic, 
and  butyric  acids  may  often  be  found  as  by-products  in  variable 
amounts.^ 

Oxydases  {Oxydizing  Enzymes). — The  most  common  example  of 
oxidation  by  means  of  bacterial  ferments  is  the  production  of  acetic  acid 

*  Buchner  und  Meisenheimer,  Ber.  d  Deut.  chem.  Gesellsch.,  xxxvi,  1903. 


THE  BIOLOGICAL  ACTIVITIES  OF  BACTERIA  51 

from  weak  solutions  of  ethyl  alcohol.  This  process,  which  is  the  basis  of 
vinegar  production,  is  universally  carried  out  by  bacterial  ferments. 
While  possessed  to  some  extent  by  a  considerable  number  of  microorgan- 
isms, acetic  acid  formation  is  a  function  pre-eminently  of  the  bacterial 
groups  described  by  Hansen,  including  ''Bacterium  aceti"  and  "Bac- 
terium pasteurianum."  To  these  two  original  groups  a  number  of  others 
have  since  been  added. 

The  organisms  are  short,  plump  bacilli,  with  a  tendency  to  chain- 
formation,  and  occasionally  showing  characteristically  swollen  centers 
and  many  irregular  involution  forms.  In  the  production  of  vinegar,  as 
generally  practiced  by  the  farmer  with  cider  or  wine,  these  bacteria 
accumulate  on  the  surface  of  the  fluid  as  a  pellicle  or  scum  which  is 
popularly  known  as  the  ''mother  of  vinegar."  Destruction  of  these 
bacteria  by  disinfectants  or  by  sterilization  with  heat  promptly  arrests 
the  process  of  vinegar  formation.  Chemically,  the  conversion  of  the 
alcohol  consists  in  a  double  oxidation  through  ethyl  aldehyde  into  acetic 
acid  as  shown  in  the  following  formulae: 

1.  C2H5  (OH)   +  O  =  CH3  (COH) 
Alcohol  Ethyl  aldehyde 

2.  CH3  (COH)   +  0  =  CH3  (COOH) 
Acetic  acid 

Alcoholic  Fermentation  {Zymase). — The  formation  of  alcohol  as  an 
end  product  of  fermentation  is  of  great  importance  in  a  number  of  the 
industries,  primarily  in  the  production  of  wine  and  beer.  AVhile  accom- 
plished by  a  number  of  bacteria,  thi^  form  of  fermentation  is  carried 
out  chiefly  by  the  yeasts. 

Expressed  in  formulae  the  simplest  varieties  of  alcoholic  fermenta- 
tion, from  mono-  and  disaccharids,  may  be  represented  as  follows: 

C«H,A=2C2H3  (OH)  +2CO2 
Dextrose  Ethyl  alcohol 

or 

C^H^^O,,  +  H2O  =  4C2H3(OH)  +  4CO2 
Saccharose  Ethyl  alcohol 

In  all  cases  the  process  may  not  be  so  simple  as  indicated  by  the  equa- 
tions, since  by-products,  such  as  higher  alcohols,  glycerin,  succinic 
and  acetic  acids,  may  often  be  found  in  small  traces  among  the  end- 
products  of  such  fermentations.    The  conditions  which  favor  alcoholic 


52  BIOLOGY    AND   TECHNIQUE 

fermentation  by  the  yeasts  are  extremely  important,  since,  upon  obser- 
servance  of  these,  depends  much  of  the  uniformity  of  result  which  is  so 
desirable  in  the  industries  mentioned  above.  The  optimum  concentra- 
tion of  sugar  for  the  production  of  the  highest  quantity  of  alcohol  is  at 
or  about  25  per  cent.  The  temperature  favoring  the  process  ranges 
about  30°  C.  Under  such  conditions  fermentation  may  continue  until 
the  alcohol  forms  almost  a  20-per-cent  solution.  Most  of  the  fermenta- 
tions important  in  the  wine,  beer,  and  spirit  industries,  take  place  under 
anaerobic  conditions,  since  the  carbon  dioxide  which  is  formed  soon 
shuts  out  any  excess  of  air. 

In  the  industrial  employment  of  yeasts  for  fermentative  purposes,  it 
is  necessary  to  work  with  specific  strains,  and  in  scientifically  conducted 
vineyards,  breweries,  and  distilleries  the  study  and  pure  cultivation  of 
the  yeasts  form  no  unimportant  part  of  the  work.  Certain  races  of  yeasts 
are  more  uniform  in  their  fermentative  powers  than  others,  and  the  by- 
products formed  by  some  races  differ  sufficiently  from  those  of  other 
races  to  cause  material  differences  in  the  resulting  substances.  In  the 
wine  industries,  the  yeasts  differ  much  from  one  another  according  to 
climatic  and  other  environmental  conditions.  In  vineyards,  natural 
inoculation  of  the  grapes  occurs  by  transportation  of  the  yeast  from 
the  soil  to  the  surface  of  the  grapes  by  wasps,  bees,  or  other  insects, 
through  whose  alimentary  canals  the  microorganisms  pass  uninjured. 
In  the  autumn  the  yeast  is  returned  to  the  soil  by  falling  berries  and 
remains  alive  in  the  upper  layers  of  the  ground  throughout  the  winter 
months.  In  actual  practice  this  natural  yeast  inoculation  is  not  de- 
pended upon,  but  pure  cultures  of  artificially  cultivated  yeasts  are 
employed  for  inoculation.  In  some  of  the  wine-growing  countries  these 
are  supplied  by  special  government  experiment  stations. 

Denitrifying  Bacteria. — Nitrogen  is  most  readily  absorbed  by  plants 
in  the  form  of  nitrates.  These  are  furnished  to  the  soil  chiefly  by  the 
proteid  decomposition  induced  by  the  proteolytic  bacterial  enzymes. 
It  is  self-evident,  therefore,  that  any  cleavage  which  reduces  nitrog- 
enous matter  beyond  the  stage  of  nitrates,  to  nitrites  and  ammonia, 
detracts  from  the  value  of  the  nitrogen  as  a  food  stuff  for  plants, 
and  the  eventual  setting  free  of  nitrogen  in  the  elementary  state  ren- 
ders it  entirely  valueless  for  any  but  the  leguminous  plants. 

Nevertheless,  this  process  of  nitrogen  waste  or  denitrification  is 
constantly  going  on  in  nature.  In  the  course  of  ordinary  decomposition, 
there  is  a  constant  reduction  of  nitrogenous  matter  to  nitrites  and  salts 
of  ammonia,  actively  taken  part  in  by  a  host  of  bacteria,  as  many  as 


THE  BIOLOGICAL   ACTIVITIES   OF    BACTERIA  53 

85  out  of  109  investigated  by  Maassen  ^  being  found  to  possess  this  power. 
This,  however,  is  not  nearly  so  harmful  a  source  of  nitrogen  waste  as 
the  process  technically  spoken  of  as  true  denitrification,  in  which 
nitrates  are  reduced,  through  nitric  and  nitrous  oxides,  to  elementary 
nitrogen. 

This  phenomenon,  more  widely  spread  among  bacteria  than  at  first 
believed,  depends  essentially  upon  simple  oxygen  extraction  from  the 
nitrates  by  the  bacteria,  and  for  this  reason  goes  on  most  actively  when 
the  supply  of  atmospheric  oxygen  is  low.  The  first  bacteria  described' 
as  possessing  this  power  of  denitrification  were  the  so-called  B.  denitri- 
ficans  I  and  II,  the  first  an  obligatory  anaerobe,  the  other  a  facultative 
aerobe.  Since  then  numerous  other  bacteria,  among  them  B.  coli  and 
B.  pyocyaneus,  have  been  shown  to  exhibit  similar  activities.  It  is 
important  agriculturally,  therefore,  to  know  that  many  species  which 
are  able  to  utilize  atmospheric  oxygen  when  supplied  with  it,  will  get 
their  oxygen  by  the  reduction  of  nitrates  and  nitrites  when  free  oxygen 
is  withheld.  It  is  thus  clear  that  a  loss  of  nitrogen  is  much  more  apt 
to  proceed  rapidly  in  manure  heaps  which  are  piled  high  and  poorly 
aerated.  There  are  other  factors,  however,  in  regard  to  the  physi- 
ology of  these  microorganisms,  which  must  be  considered  for  practical 
purposes. 

In  order  that  these  bacteria  may  develop  their  denitrifying  powers 
to  the  best  advantage,  it  is  necessary  to  supply  them  with  some  carbon 
compound  which  is  easily  absorbed  by  them.  This,  in  decomposing 
material,  is  furnished  by  the  products  of  the  carbohydrate  cleavage 
going  on  side  by  side  with  the  proteolytic  processes.  It  is  still  more 
or  less  an  open  question  whether  the  facilitation  of  denitrification 
brought  about  in  manure  heaps  by  the  presence  of  hay  and  straw  is  due 
to  the  carbon  furnished  by  these  materials,  or  whether  it  is  due  to  the 
fact  that  bacilli  of  this  group  are  apt  to  adhere  to  the  straw  which  acts 
in  that  case  as  a  means  of  inoculation. 

The  actual  danger  of  nitrogen  depletion  of  the  soil  by  denitrifying 
processes  is  probably  much  less  threatening  than  was  formerly  supposed; 
for,  in  the  first  place,  the  conditions  for  complete  denitrification  are 
much  more  perfect  in  the  experiment  than  they  ever  can  be  in  nature, 
and  the  nitrifying  processes  going  on  side  by  side  with  denitrification 
make  up  for  much  of  the  loss  sustained. 


Mm99en,  Arb,  a,  d.  kais.  Gesundheitsamt,  1,  xxviii,  1901, 


54  BIOLOGY   AND   TECHNIQUE 


ANABOLIC    OR    SYNTHETIC    ACTIVITIES    OF    BACTERIA 

Nitrogen  Fixation  by  Bacteria. — The  constant  withdrawal  of  nitroge- 
nous substances  from  the  soil  by  innumerable  plants  would  soon  lead  to 
total  depletion  were  it  not  for  certain  forces  continually  at  work  re- 
plenishing the  supply  out  of  the  large  store  of  free  nitrogen  in  the  atmos- 
phere. This  important  function  of  returning  nitrogen  to  the  soil  in 
suitable  form  for  consumption  by  the  plants  is  performed  largely  by 
bacteria. 

It  is  well  known  that  specimens  of  agricultural  soil  when  allowed  to 
stand  for  any  length  of  time  without  further  interference  will  increase 
in  nitrogenous  content,  but  that  similar  specimens,  if  sterilized,  will 
show  no  such  increase.^  The  obvious  conclusion  to  be  drawn  from  this 
phenomenon  is  that  some  living  factor  in  the  unsterilized  soil  has  aided 
in  increasing  the  nitrogen  supply.  Light  was  thrown  upon  this  problem 
when  Winogradsky,^  in  1893,  discovered  a  microorganism  in  soil  which 
possessed  the  power  of  assimilating  large  quantities  of  nitrogen  from 
the  air.  This  bacterium,  which  he  named  "  Clostridium  Pasteurianum," 
is  an  obligatory  anaerobe  which  in  nature  always  occurs  in  symbiosis 
with  two  other  facultatively  anaerobic  microorganisms.  In  sym- 
biosis with  these,  it  can  be  cultivated  under  aerobic  conditions  and  thus 
grows  readily  in  the  upper  well-aerated  la3^ers  of  the  soil. 

Although,  until  now,  no  other  bacteria  with  equally  well-developed 
nitrogen-fixing  powers  have  been  discovered,  yet  it  is  more  than  likely 
that  Clostridium  Pasteurianum  is  not  the  only  microorganism  endowed 
with  this  function.  In  fact,  Penicillium  glaucum  and  Aspergillus  niger, 
two  molds,  and  two  other  bacteria  described  by  Winogradsky,  have  been 
shown  to  possess  this  power  slightly,  but  in  an  incomparably  less  marked 
degree  than  Clostridium  Pasteurianum.^  According  to  the  calculations 
of  Sachse,*  unsterilized  soil  may,  under  experimental  conditions,  gain 
as  much  as  25  milligrams  of  nitrogen  in  a  season,  a  statement  which 
permits  the  calculation  of  a  gain  of  twelve  kilograms  of  nitrogen  per 
acre  annually.^  It  is  very  unlikely,  however,  that  such  gains  actually 
occur  in  nature,  where  nitrogen-fixation  and  nitrogen-loss  usually 
occur  side  by  side. 

1  Berthelot,  Compt.  rend,  de  la  soc.  de  bioL,  cxvi,  1893. 

2  Winogradsky,  Compt.  rend,  de  la  soc.  de  biol.,  cxvi,  1893,  ibid.,  t.  cxviii,  1894. 

3  Tacke,  Landwirtsch.  Jahresber.,  xviii,  1889. 
a^ac/ise,  "Agr.  Chem.,"  1883. 

«  Pfeffer,  Pflugers  Physiologie,  p.  395. 


THE    BIOLOGICAL   ACTIVITIES    OF    BACTERIA  55 

Agriculturally  of  even  greater  importance  than  the  free  nitrogen- 
fixing  bacteria  of  the  soil  are  the  bacteria  found  in  the  root  tubercles  of 
a  class  of  plants  known  as  "leguminosse."  It  has  long  been  known  that 
this  class  of  plants,  including  clover,  peas,  beans,  vetch,  etc., not  only  does 
not  withdraw  nitrogen  from  the  soil,  but  rather  tends  to  enrich  it.  Upon 
this  knowledge  has  depended  the  well-known  method  of  alternation  of 
crops  employed  by  farmers  the  world  over.  The  actual  reason  for  the 
beneficial  influence  of  the  leguminosae,  however,  was  not  known  until 
1887,  when  Hellriegel  and  Wilfarth  ^  succeeded  in  demonstrating  that 
the  nitrogen-accumulation  was  directly  related  to  the  root  tubercles  of 
the  plants,  and  to  the  bacteria  contained  within  them. 

These  tubercles,  which  are  extremely  numerous — as  many  as  a 
thousand  sometimes  occurring  upon  one  and  the  same  plant — are  formed 
by  the  infection  of  the  roots  with  bacteria  which  probably  enter  through 
the  delicate  root-hairs.  They  Vjary  in  size,  are  usually  situated  near  the 
main  root-stem,  and,  in  appearance,  are  not  unlike  fungus  growths. 
Their  development  is  in  many  respects  comparable  to  the  develop- 
ment of  inflammatory  granulations  in  animals  after  infection,  inas- 
much as  the  formation  of  the  tubercle  is  largely  due  to  a  reactionary 
hyperplasia  of  the  plant  tissues  themselves.  They  appear  upon  the 
seedlings  within  the  first  few  weeks  of  their  growth  as  small  pink 
nodules,  and  enlarge  rapidly  as  the  plant  grows.  At  the  same  time, 
later  in  the  season,  when  the  plants  bear  fruit,  the  root  tubercles  begin 
to  shrink  and  crack.  When  the  crops  are  harvested,  the  tubercles  with 
the  root  remain,  rot  in  the  ground,  and  re-infect  the  soil. 

Histologically  the  tubercles  are  seen  to  consist  of  large  root  cells 
which  are  densely  crowded  with  microorganisms. 

The  microorganism  itself,  "Bacillus  radicicola,"  was  first  observed 
within  the  tubercles  by  Woronin  ^  in  1866.  The  bacilH  are  large,  slender, 
and  actively  motile  during  the  early  development  of  the  tubercles,  but 
in  the  later  stages  assume  a  number  of  characteristic  involution  forms, 
commonly  spoken  of  as  "bacteroids."  They  become  swollen,  T  and  Y 
shaped,  or  branching  and  threadlike.  Their  isolation  from  the  root 
tubercles  usually  presents  Httle  difficulty,  since  they  grow  readily  upon 
gelatin  and  agar  under  strictly  aerobic  conditions.  On  the  artificial 
media  the  bacillary  form  is  usually  well  retained,  involution  forms 
appearing  only  upon  old  cultures. 


1  HeUriegel  und  WilfaHh,  Cent.  f.  Bakt.,  1887. 

2  Woronin,  Bot.  Zeit.,  xxiv,  1866. 


56  BIOLOGY   AND   TECHNIQUE 

The  classical  experiments  of  Hellriegel  and  Wilfarth  conclusively 
demonstrated  the  important  relation  of  these  tubercle-bacteria  to  nitro- 
gen assimilation  by  the  leguminosae. 

These  observers  cultivated  various  members  of  this  group  of  plants 
upon  nitrogen-free  soil — sand — and  prevented  the  formation  of  root 
tubercles  in  some,  by  sterilization  of  the  sand,  while  in  others  they 
encouraged  tubercle  formation  by  inoculation.  An  example  of  their 
results  may  be  given  as  follows:  ^ 

Lupinus  luteus  was  cultivated  upon  sterilized  sand.  Some  of  the 
pots  were  inoculated  with  B.  radicicola,  others  were  kept  sterile.  Com- 
parative analyses  were  made  of  the  plants  grown  in  the  different  pots 
with  the  following  striking  result: 


Root  tubercles 
present 


No  root  tubercles , 


IVllJ.^      1COU.XU 

• 

N 

.  added  in 

seed, 

Harvested 

soil,  and  soil- 

Gain  or 

dry  weight 

N. 

present 

extract 

loss  of  N. 

(a)    38.919 

.998 

.022 

+  .975 

(b)   33.755 

.981 

.023 

-f.958 

(c)     0.989 

.016 

.020 

—  .004 

(d)     0.828 

.011 

.022 

—  .009 

The  great  importance  of  this  process  in  agriculture  is  demonstrated, 
furthermore,  by  a  comparison  made  by  the  same  observers  between  a 
legume,  the  pea,  and  one  of  the  common  nitrogen-consuming  crops,  oats.^ 

Nitrogen  contents  Nitrogen  contents 

of  seed  and  soil.                             of  crop.  Gain  or  loss. 

Oats  0.027  grams                        0.007  grains  —.020 

Peas  0.038     "                            0.459      "  +.421 

Exactly  what  the  process  is  by  which  the  bacteria  supply  nitrogen  to 
the  plant  is  as  yet  uncertain.  Although  the  degenerating  bacteroids  in 
old  nodules  are  bodily  absorbed  by  the  plant,  this  can  not  be  con- 
ceived as  the  only  method  of  supply,  since  the  total  nitrogen  gain  many 
times  exceeds  the  total  weight  of  bacteria  in  the  nodules.  It  is  probable 
that  the  microorganisms  during  life  take  up  atmospheric  nitrogen  and 
secrete  a  nitrogenous  substance  which  is  absorbed  by  the  plant  cells. 

Although  formerly  the  relationship  between  plant  and  bacterium 
was  regarded  as  one  of  symbiosis  and  of  mutual  benefit,  the  opinions  as 
to  this  subject  show  wide  divergence.  While,  according  to  some  authors, 
the  entrance  of  the  bacteria  into  the  plants  is  regarded  as  a  true  in- 
fection against  which  the  plant  offers  at  first  a  determined  opposition  as 
evidenced  by  tissue  reactions,  other  observers,  notably  A.  Fischer,  regard 

1  Pfeffer,  "  Planzenphysiologie,"  Leipzig,  1897. 

2  Hellriegel  und  Wilfarth,  Zeit.  d.  Ver.  f.  d.  Riibenzucker  Industrie,  1888.  Quote/ 
from  Fischer,  "  Vorles.  uber  die  Bakt.,"  Jena,  1903 


THE   BIQL.OGICAL   ACTIVITIES   OF   BACTERIA  57 

the  plant  as  a  parasite  upon  the  bacteria,  in  that  it  derives  the  sole 
benefit  from  the  relationship  and  eventually  bodily  consumes  its  host. 
Nitrif3ring  Bacteria. — A  process  diametrically  opposed  in  its  chem- 
istry to  denitrification  and  reduction  is  that  which  brings  about  an 
oxidation  of  ammonia  to  nitrites  and  nitrates.  The  actual  increase 
of  nitrates  in  soil  allowed  to  stand  for  any  length  of  time  and  examined 
from  time  to  time  has  been  a  well-established  fact  for  many  years;  but 
it  was  believed  until  a  comparatively  short  time  ago  that  this  increase 
was  due  to  a  simple  chemical  oxidation  of  ammonia  by  atmospheric  oxy- 
gen. The  dependence  of  nitrification  upon  the  presence  of  living  organ- 
isms was  finally  proved  by  Muntz  and  Schlossing  ^  in  1887,  who  demon- 
strated that  nitrification  was  abruptly  stopped  when  the  soil  was 
sterilized  by  heat  or  antiseptics.  It  remained,  however,  to  isolate  and 
identify  the  organisms  which  brought  about  this  ammonia  oxidation. 
This  last  step  in  our  knowledge  of  nitrification  was  taken  in  1890,  by 
Winogradsky.  Winogradsky  ^  found  that  the  failures  experienced  by 
others  who  had  attempted  to  isolate  nitrifying  bacteria  were  due  to  the 
fact  that  they  had  used  the  common  culture  media  largely  made  up  of 
organic  substances.  By  using  culture  media  containing  no  organic 
matter  Winogradsky  succeeded  in  isolating  free  from  the  soil,  bacteria 
which  have  since  that  time  been  confirmed  as  being  the  causative  factors 
in  nitrification.  During  his  first  experiments  this  author  observed  that 
in  some  of  his  cultures  the  oxidation  of  ammonia  went  only  as  far  as  the 
stage  of  nitrite  formation,  while  in  others  complete  oxidation  to  nitrates 
took  place.  Following  the  clews  indicated  by  this  discrepancy,  he 
finally  succeeded  in  demonstrating  that  nitrification  is  a  double  process 
in  which  two  entirely  different  varieties  of  microorganisms  take  part, 
the  one  capable  of  oxidizing  ammonia  to  nitrites,  the  other  continuing 
the  process  and  converting  the  nitrites  to  nitrates.  The  nitrite-forming 
bacteria  discovered  by  Winogradsky,  and  named  Nitromonas  or  Nitro- 
somonas,  are  easily  cultivated  upon  aqueous  solutions  containing  am- 
monia, potassium  sulphate,  and  magnesium  carbonate.  According  to 
their  discoverer  they  develop  within  a  week  in  this  medium  as  a  gelat- 
inous sediment.  After  further  growth  this  sediment  seems  to  break 
up  and  the  bacteria  appear  as  oval  bodies,  which  swim  actively  about 
and  develop  flagella  at  one  end.  Upon  the  solid  media  in  ordinary  use 
they  can  not  be  cultivated.    Special  solid  media  suitable  for  their  cul- 


»  Muntz  und  Schlossing,  Compt.  rend,  de  I'acad.  des  sciences,  1887* 
2  Winogradsky,  Ann.  Past.  Inst.,  iv  and  v,  1890,  1891. 


58  BIOLOGY   AND    TECHNIQUE 

tivation  and  composed  of  silicic  acid  and  inorganic  salts  have  been 
described  by  Winogradsky  and  by  Omeliansky/ 

Other  nitrite-forming  bacteria  have  since  been  described  by  various 
observers,  all  of  them  more  or  less  limited  to  definite  localities.  Some 
of  these  are  similar  to  nitrosomonas  in  that  they  exhibit  the  flagellated^ 
actively  motile  stage.    In  others  this  stage  is  absent. 

The  nitrite-forming  bacteria,  apart  from  their  great  agricultural  im- 
portance, claim  our  attention  because  of  their  unique  position  in  rela- 
tion to  the  animal  and  vegetable  kingdoms.  Extremely  sensitive  to 
the  presence  of  organic  compounds,  they  are  able  to  grow  and  develop 
only  upon  media  containing  nothing  but  inorganic  material;  and  this 
entirely  without  the  aid  of  any  substances  comparable  to  the  chlorophyll 
of  the  green  plants.  The  source  of  energy  from  which  this  particular 
class  of  bacteria  derive  the  power  of  building  up  organic  compounds 
from  simple  substances  is  to  some  extent  a  mystery.  The  carbon 
which  they  unquestionably  require  for  the  building  up  of  organic  mate- 
rial may  be,  as  Winogradsky  believed,  derived  to  a  certain  extent  from 
ammonium  carbonate.  But  it  is  also  quite  certain  that  they  are  capable 
of  utilizing  directly  atmospheric  CO  2.  In  the  absence  of  chlorophyll 
or  of  any  highly  organized  chemical  compound,  it  seems  likely  that 
the  energy  necessary  for  the  utilization  of  the  carbon  obtained  in  this 
simple  form  is  derived  from  the  oxidation  of  ammonia  during  the  proc- 
ess of  nitrification. 

The  conversion  of  nitrites  into  nitrates  is  carried  on  by  other  species 
of  bacteria  also  discovered  by  Winogradsky.  These  bacteria  are  much 
more  generally  distributed  than  nitrosomonas  and  probably  include  a 
number  of  varieties.  The  organism  described  by  Winogradsky  is  an 
extreinely  small  bacillus  with  pointed  ends.  Capsules  have  occasionally 
been  made  out.  It  may  be  cultivated  upon  aqueous  solutions  con- 
taining: 

Sod.  nitrite 1  per  cent. 

Potass,  phosphate : 05     "       " 

Magnesium  sulph 03      "       " 

Sodium  carbonate 1        "       " 

Ferrous  sulphate 04  "       " 

The  development  of  the  organism  is  slow  and  sparse,  and  is  directly 
inhibited  by  the  presence  of  organic  matter.  It  is  strongly  inhibited  by 
the  presence  of  ammonia. 

The  Liberation  of  Energy  by  Bacteria.— Like  all  other  living  beings, 

1  Omeliansky,  Cent.  f.  Bakt.,  II,  5,  1899. 


THE  BIOLOGICAL  ACTIVITIES  OP  BACTERIA  59 

bacteria  in  their  metabolic  processes  liberate  energy.  It  has  been 
shown  by  several  observers  that  slight  quantities  of  heat  are  given 
off  from  actively  growing  cultures.  The  functions,  furthermore,  of 
reproduction,  motility,  and  enzyme  formation  may  be  looked  upon  as 
forms  of  energy  liberation.  In  addition  to  this,  certain  bacteria  have 
been  observed  which  may  liberate  energy  in  the  form  of  light. 

Light  Production  by  Bacteria. — ^The  production  of  light  by  bacteria 
is  a  power  possessed  chiefly  by  certain  species  inhabiting  salt  water. 
Thus,  much  of  the  phosphorescence  observed  at  sea,  though  more  fre- 
quently due  to  Medusa  and  other  invertebrate  animals,  is  caused  by 
these  bacteria.  Numerous  species  which  produce  this  phenomenon 
have  been  isolated,  too  many,  and  too  unimportant,  to  be  individually 
described.  All  of  them  are  aerobes  and  require  highly  complex  food 
stuffs.  They  are  closely  allied  to  the  putrefactive  bacteria,  and  in 
the  sea  are  usually  found  upon  rotting  animal  matter.^  The  production 
of  light  seems  directly  dependent  upon  the  free  access  of  oxygen,  since 
no  light  appears  under  anaerobic  conditions.  Their  luminous  quality, 
moreover,  is  not  a  true  phosphorescence,  in  that  it  does  not  depend 
upon  previous  illumination  and  develops  as  well  in  cultures  kept  in  the 
dark  as  in  those  which  have  been  exposed  to  light. ^ 

The  Formation  of  Pigment  by  Bacteria  {Chromobacteria) . — A  large 
number  of  bacteria,  when  cultivated  upon  suitable  media,  give  rise  to 
characteristic  colors  which  are  valuable  as  marks  of  differentiation. 
For  each  species,  the  color  is  usually  constant,  depending,  to  a  certain 
extent,  upon  the  conditions  of  cultivation.  In  only  a  few  of  the 
pigmented  bacteria  is  the  pigment  contained  within  the  cell  body,  and 
in  only  one  variety,  the  sulphur  bacteria,  does  the  pigment  appear  to 
hold  any  distinct  relationship  to  nutrition.  In  most  cases,  the  coloring 
matter  is  found  to  be  deposited  in  small  intercellular  granules  or  globules. 
The  absence  of  any  relationship  of  the  pigment  to  sunlight,  as  is  the  case 
with  the  chlorophyll  of  the  green  plants,  is  indicated  by  the  fact  that 
most  of  the  chromobacteria  thrive  and  produce  pigment  equally  well  in 
the  dark  as  they  do  in  the  presence  of  light.  Among  the  most  common 
of  the  pigment  bacteria  met  with  in  bacteriological  work  are  Staphy- 
lococcus pyogenes  aureus,  Bacillus  pyocyaneus.  Bacillus  prodigiosus, 
and  some  of  the  green  fluorescent  bacteria  frequently  found  in  feces. 

The  chemical  nature  of  these  pigments  has  been  investigated  quite 
thoroughly  and  it  has  been  shown  that  they  vary  in  composition. 

1  Pfliiger's  Arch.  f.  Phys.,  xi,  1875.       2  Fisclwr,  Cent.  f.  Bakt.,  iii,  1888. 


^  BIOLOGY    AND   TECHNlQOT 

Some  of  the  pigments,  like  that  of  Staphylococcus  aureus,  are  probably 
non-proteid  and  of  a  fatty  nature.*  They  are  insoluble  in  water  but 
soluble  in  alcohol,  ether,  and  chloroform.  Because  of  their  probable 
composition,  they  have  been  spoken  of  as  ''lipochromes."  Other 
pigments,  like  the  pyocyanin,  which  lends  the  green  color  to  cultures 
of  Bacillus  pyocyaneus,  are  water  soluble  and  are  probably  of  proteid 
composition.  Pyocyanin  may  be  crystallized  out  of  aqueous  solu- 
tion in  the  form  of  fine  needles.  The  crystals  may  be  redissolved  in 
chloroform.  Aqueous  solutions  retain  their  color.  Solutions  in  chloro- 
form, however,  are  changed  gradually  to  yellow. 

The  power  of  pigment  production  of  various  bacteria  depends  in 
each  case  upon  cultural  conditions.  In  most  cases,  this  simply  signifies 
that  pigment  is  produced  only  when  the  microorganism,  finding  the  most 
favorable  environmental  conditions,  is  enabled  to  develop  all  its  func- 
tions to  their  fullest  extent.  Thus,  a  too  high  acidity  or  alkalinity  of 
the  culture  medium  may  inhibit  pigment  formation.  Oxygen  is  neces- 
sary for  the  production  of  color  in  some  bacteria,  since  the  bacteria  them- 
selves often  produce  the  pigment  only  as  a  leuko-body  which  is  then 
oxydized  into  the  pigment  proper.  A  notable  example  of  this  is  the  pig- 
ment of  B.  pyocyaneus.  In  other  cases,  temperature  plays  an  impor- 
tant role  in  influencing  color  production.  Thus,  Bacillus  prodigiosus 
refuses  to  produce  its  pigment  when  growing  in  the  incubator.  By 
persistent  cultivation  in  an  unfavorable  environment,  colored  cultures 
may  lose  their  power  of  pigment  production. 

Sulphur  Bacteria. — Wherever  the  decomposition  of  organic  matter 
gives  rise  to  the  formation  of  Hj  S,  in  cess-pools,  in  ditches,  at  the  bottom 
of  the  sea,  and  in  stagnant  ponds,  there  is  found  a  curiously  interesting 
group  of  microorganisms,  the  so-called  sulphur  or  thiobacteria.  Red, 
purple,  and  colorless,  these  bacteria  all  possess  the  power  of  utilizing 
sulphuretted  hydrogen  and  by  its  oxidation  into  free  sulphur  obtain 
the  energy  necessary  for  their  metabolic  processes.  The  colorless  sul- 
phur bacteria,  the  Beggiatoa  and  Thiothrices,  usually  appear  as  threads 
or  chains  which,  in  media  containing  sufficient  H3  S,  are  usually  well- 
stocked  with  minute  globules  of  sulphur.  If  found  upon  decomposing 
organic  matter,  they  often  cover  this  as  a  grayish  mold-like  layer. 
The  red  sulphur  bacteria,  of  which  numerous  species  have  been  described 
by  Winogradsky,  may  appear  as  actively  motile  spirilla  (Thiospirillum) 
or  as  short,  thick  bacillary  forms. 

» Schroeter.  Gent.  f.  Bakt..  xviii.  1895. 


THE   BIOLOGICAL    ACTIVITIES    OF    BACTERIA  61 

The  physiology  of  all  the  sulphur  bacteria,  and  especially  of  the 
colored  varieties,  is  of  the  greatest  interest  in  that  these  microorganisms 
are  among  the  few  members  of  the  bacterial  group  which  behave  meta- 
bolically  like  the  green  plants.  The  higher  organic  substances  play  lit- 
tle or  no  part  in  the  nutrition  of  these  microorganisms.  Strictly  aerobic, 
the  colorless  thiobacteria  are  independent  of  sunlight,  while  the  red  and 
purple  varieties  exhibit  their  physiological  dependence  upon  light  by 
accumulating  under  natural  conditions  in  well-lighted  spots.  Both 
varieties  possess  equally  the  power  of  oxidizing  sulphuretted  hydrogen 
as  a  source  of  energy.  The  sulphur  is  then  stored  as  elemental  sulphur 
within  the  bacterial  body  and  when  a  lack  of  food  stuffs  sets  in,  the 
store  of  sulphur  can  be  further  oxidized  into  sulphurous  or  sulphuric 
anhydrides.  With  this  sole  source  of  energy,  these  bacteria  are  capable 
of  flourishing  aerobic  ally,  while  an  absence  of  HjS,  even  in  the  presence 
of  organic  food  stuffs,  leads  to  a  rapid  disappearance  of  their  sulphur 
contents  and  an  inability  to  develop. 

In  the  case  of  the  colored  thiobacteria,  the  red  pigment  appears  to 
fulfil,  to  some  extent,  a  function  comparable  to  that  of  the  chlorophyll 
of  the  green  plants. 

Engelmann,*  who  has  studied  this  pigment  spectroscopically,  has 
found  that  besides  absorbing  the  red  spectral  rays  there  is  an  absorption 
of  rays  on  the  ultra-red  end  of  the  spectrum.  The  absorption  of  the 
red  rays  between  the  lines  B  and  C  of  the  spectrum,  and  of  violet  rays 
at  the  line  F,  is  the  same  as  that  of  the  absorption  spectrum  of 
chlorophyll,  and  it  is  in  the  zone  of  these  rays  that  the  physiological 
effects  of  chlorophyll  are  most  active.  In  addition  to  these  absorp- 
tion bands,  the  bacteriopurpurin  of  the  red  sulphur  bacteria  shows 
absorption  of  the  invisible  ultra  red  rays  of  the  spectrum. 

Engelmann,  with  a  microspectroscope,  projected  a  spectrum  into 
a  miscroscopic  field  in  which  green  algae  or,  in  the  case  under  discussion, 
red  sulphur  bacteria  had  been  placed.  Other  sources  of  light  were,  of 
course,  excluded.  By  adding  emulsions  of  strictly  aerobic  bacteria  to 
such  preparations,  an  accumulation  of  microorganisms  was  observed 
at  those  points  in  the  spectrum  at  which  most  oxygen  was  liberated.  In 
the  case  both  of  chlorophyll  and  of  the  red  sulphur  bacteria  such  areas 
of  bacterial  accumulation  (in  oxygen  Hberation)  occurred  in  the  zones 
of  the  absorption  bands  mentioned  above. 


Engelmann,  Bot.  Zeit.,  1888. 


CHAPTER  V 
THE   DESTRUCTION    OF   BACTERIA 

GENERAL    CONSIDERATIONS 

No  branch  of  bacteriology  has  been  more  fruitful  in  practical  appli- 
cation than  that  which  deals  with  the  factors  which  bring  about  the 
destruction  of  microorganisms.  Upon  the  study  of  this  branch  has 
depended  the  growth  and  the  development  of  modem  surgery. 

The  agents  which  affect  bacteria  injuriously  are  many,  and  are  both 
physical  and  chemical  in  nature. 

When  a  procedure  completely  destroys  bacterial  life  it  is  spoken  of  as 
sterilization  or  disinfection,  the  term  disinfection  being  employed  more 
especially  to  designate  the  use  of  chemical  agents.  When  the  procedure 
destroys  vegetative  forms  only,  leaving  the  more  resistant  spores  un- 
injured, it  is  spoken  of  as  "incomplete  sterilization."  When  an  agent, 
on  the  other  hand,  does  not  actually  kill  the  microorganisms,  but  merely 
inhibits  their  growth  and  multiphcation,  it  is  spoken  of  as  an  antiseptic. 
The  term  deodorant  is  indiscriminately  applied  to  substances  which 
mask  or  destroy  offensive  odors,  and  may  or  may  not  possess  disinfectant 
or  antiseptic  value.  Some  deodorants  act  chemically  on  the  noxious 
gases,  destroying  them. 

PHYSICAL   AGENTS    INJURIOUS    TO   BACTERIA 

The  principal  physical  agents  which  may  exert  deleterious  action 
upon  bacteria  are:  drying,  light,  electricity,  and  heat. 

Drying. — Complete  desiccation  eventually  destroys  most  of  the  path- 
ogenic bacteria,  yet  great  differences  in  resistance  to  this  condition  are 
shown  by  various  microorganisms.  Ficker,^  who  has  made  a  systematic 
study  of  the  influence  of  complete  drying  upon  bacteria,  concludes  that 
the  resistance  of  bacteria  to  desiccation  is  influenced  by  the  age  of  the 
culture  investigated,  the  rapidity  with  which  the  withdrawal  of  moisture 

1  Ficker,  Zeit.  f.  Hyg.,  xxix,  1896. 
62 


THE  DESTRUCTION  OF  BACTERIA  63 

is  accomplished,  and  the  temperature  at  which  the  process  takes  place. 
Microorganisms  like  the  gonococcus  and  the  Pfeiffer  bacillus,  are 
destroyed  by  drying  within  a  few  hours.  The  cholera  vibrio  dried  upon  a 
coverslip  was  found  by  Koch  ^  to  be  killed  within  four  hours;  by  Burck- 
holtz,^  to  survive  about  twenty-four  hours.'  The  spore-forms  of  bacteria 
are  infinitely  more  resistant  to  this  influence  than  are  the  vegetative 
forms,  though  they  may  be  destroyed  by  rapid  and  complete  drying  in 
a  desiccator. 

It   is   self-evident   that   many   discrepancies   in   the   experimental 
results  of  various  authors  may  depend  upon  the  technique  of  investiga- , 
tion,  since  the  degree  of  drying  attained  depends  intimately  upon  the 
thickness  and  consistence  of  the  material  investigated,  and  upon  the 
methods  employed  for  desiccation. 

Light. — Direct  sunlight  is  a  powerful  germicide  for  all  bacteria  except 
a  limited  number  of  species  like  the  thio-or  sulphur  bacteria,  which 
utilize  sunlight  for  their  metabolic  processes  as  do  the  green  plants. 

Koch  ^  has  shown  that  exposure  to  sunlight  will  destroy  the  tubercle 
bacillus  within  two  hours  or  less,  the  time  depending  upon  the  thick- 
ness of  the  exposed  layers  and  the  material  surrounding  the  bacilli. 
Confirmatory  researches  have  been  published  by  Mignesco  *  and  others. 
The  powerful  disinfecting  influence  of  sunlight  upon  bacteria  suspended 
in  water  has  been  shown  by  Buchner.^  Observations  in  regard  to  the 
influence  of  sunlight  upon  anthrax  spores  have  been  made  by  Arloing,^ 
and  similar  observations  upon  a  number  of  other  microorganisms  have 
been  carried  out  by  Dieudonne,  Janowski,  v.  Esmarch,  and  many 
others.  All  these  observers,  while  differing  somewhat  as  to  the  time 
necessary  for  bacterial  destruction^  agree  in  finding  definite  and  pow- 
erful bactericidal  action  of  sunlight.  Diffuse  light,  of  course,  is  less 
active  than  direct  sunlight.  According  to  Buchner,  typhoid  bacilli  are 
inhibited  by  direct  sunhght  in  one  and  one-half  hours,  by  diffuse  light 
in  five  hours.  A  remarkable  statement  is  made  by  Arloing,  who  claims 
to  have  found  that  anthrax  spores  are  more  quickly  destroyed  by 
direct  sunlight  than  are  the  vegetative  cells.  This  fact  would  call  for 
further  confirmation. 

» Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  iii,  1887. 

2  Burkholtz,  Arb.  a.  d.  kais.  Gesundheitsamt,  v,  1889. 

3  Koch,  X  Intemat.  Med.  Congress,  Berlin,  1890. 
*  Mignesco,  Arch.  f.  Hyg.,  xxv,  1896. 

6  Buchner,  Cent.  f.  Bakt.,  I,  xi,  1892. 

«  Arloing,  Compt.  rend,  de  I'acad.  d.  scL,  c,  1885, 


64  BIOLOGY   AND   TECHNIQUE 

It  has  been  shown  by  various  authors  that  the  influence  of  sunlight  is 
not  to  be  attributed  in  anyway  to  temperature,  nor  always  to  a  direct 
action  of  the  light  upon  the  bacteria,  but  depends  largely  upon  photo- 
chemical changes  produced  by  the  light  rays  in  the  media.  Richardson  ^ 
and  Dieudonne  ^  conclude  that  under  ordinary  aerobic  conditions  in  fluid 
environment  peroxide  of  hydrogen  is  formed  under  the  influence  of  light. 
Novy  and  Freer  ^  believe  that  the  bactericidal  efi^ects  in  fluids  noticed 
as  a  result  of  exposure  to  light  are  too  strong  to  be  explained  by  the 
formation  of  small  quantities  of  peroxide  of  hydrogen,  and  attribute 
this  action  to  organic  peroxides  formed  under  the  described  conditions, 
such  as  the  peroxides  of  diacetyl,  benzoylacetyl,  and  others.  These 
views  are  somewhat  strengthened  by  the  fact  that  exclusion  of  oxygen 
from  media  markedly  diminishes  the  bactericidal  power  of  light.^  That 
the  photochemical  changes  alone,  however,  do  not  explain  this  action 
follows  from  the  fact  that  dried  bacteria,  not  surrounded  by  media,  are 
subject  to  a  similar  action.^ 

In  analyzing  sunlight  in  regard  to  its  bactericidal  power,  it  has  been 
found  by  various  observers  that  the  most  powerful  action  is  exerted  by 
the  ultraviolet  spectral  rays,  whereas  the  yellow,  red,  and  ultra-red  rays 
are  practically  innocuous.^ 

It  is  of  importance  to  note  that  sunlight  has  been  found  also  to  have 
a  strong  attenuating  influence  ^  upon  some  bacterial  poisons,  as  shown 
by  the  experiments  of  Ferri  and  Celli  upon  tetanus  toxin. 

Electric  light  exerts  a  distinct  bactericidal  action  when  applied  in 
strengths  of  800  to  900  candle  power  for  seven  or  eight  hours.^ 

Rontgen  or  a:-rays  are  said  by  Zeit,^  Blaise  ^^  and  Sambac,  and  others 
to  be  without  appreciable  geritiicidal  power.  Rieder,^^  on  the  other 
hand,  has  reported  definite  inhibition  of  bacterial  growth  after  exposures 
of  half  an  hour  to  a;-rays. 


1  Richardson,  Jour.  Chem.  Soc,  i,  1893,  Ref.  Deut.  chem.  Gesells,,  xxvi. 

2  Dievdonne,  loc.  cit. 

3  Novy  and  Freer,  3d  Ann.  Meeting  Assn.  Amer.  Bacteriologists,  Chicago,  1901. 
*  Roux,  Ann.  Inst.  Past.,  ix,  1887. 

'^  Dieudonn6,  loc.  cit. 

8  Ward,  Proc.  Royal  Soc,  52,  1893. 

.7  Fern  and  Celli,  Cent.  f.  Bakt.,  I,  xii,  1892. 

8  Dieudonne,  loc.  cit. 

^Zeit,  Jour.  Amer.  Med.  Assn.,  xxxvii,  1901. 

1°  Blaise  and  Sambac,  Compt.  rend,  de  la  soc.  de  biol.,  1896. 

"  Rieder,  Miinch.  med.  Woch.,  1898. 


THE   DESTRUCTION    OF   BACTERIA  65 

Radium  rays  have  a  distinct  inhibitory  and  even  bactericidal 
power  when  applied  at  distances  of  a  few  centimeters  for  several 
hours.  ^ 

Electricity. — If  we  exclude  the  indirect  actions  of  heat  and  electro- 
lysis, it  can  hardly  be  said  that  the  direct  bactericidal  action  of  electric 
currents  has  been  satisfactorily  demonstrated.  Such  action,  however, 
has  been  claimed  by  d'Arsonville  and  Charrin,^  and  by  Spilker  and 
Gottstein.3 

Heat. — ^The  most  widely  applicable  and  efficient  physical  agent  for 
sterilization  is  heat. 

The  dependence  of  bacteria  for  growth  and  vitality  upon  the  main- 
tenance of  a  proper  temperature  in  their  environment,  and  the  ranges 
of  variation  within  which  bacteria  may  thrive,  have  been  discussed  in  a 
preceding  section,  in  which  a  table  of  so-called  "  thermal  death  points  " 
has  been  given.  In  the  method  of  expressing  these  values  it  was  seen 
that  tv/o  elements  entered  into  the  destruction  of  bacteria  by  heat, 
namely,  that  of  the  degree  of  temperature  which  is  applied,  and  that  of 
the  time  of  application. 

The  prolonged  application  of  moderately  high  temperatures,  in  other 
words,  may  in  certain  instances,  accomplish  the  same  result  as  the  brief 
use  of  extremely  high  ones.  In  general,  the  death  of  bacteria  following 
prolonged  exposure  to  temperatures  but  slightly  exceeding  the  optimum 
is  due  to  the  inability  of  the  anabolic  processes  to  keep  pace  with  the 
accelerated  katabolic  processes,  gradual  attenuation  resulting  in  death. 
At  somewhat  higher  temperatures  death  results  from  coagulation  of 
the  bacterial  protoplasm,  and  at  still  higher  degrees  of  heat,  applied  in 
the  dry  form,  direct  burning  of  the  bacteria  may  be  the  cause  of  their 
destruction.' 

Heat  may  be  applied  in  the  form  of  dry  heat  or  as  moist  heat,  these 
methods  being  of  great  practical  value,  but  differently  applicable  ac- 
cording to  the  nature  of  the  materials  to  be  sterilized.  The  two  methods, 
moreover,  show  a  marked  difference  in  efficiency,  temperature  for  tem- 
perature. For  the  recognition  of  this  fact  we  are  largely  indebted  to  the 
early  researches  of  Koch  and  Wolff hiigel,"  and  of  Koch,  Gaffky,  and 
Loeffler.^ 

1  Personal  observations, 

2  D'Arsonville  and  Charrin,  Compt.  rend,  de  la  soc.  de  biol. 

3  Sjyilker  and  Gottstein,  Cent.  f.  Bakt.,  I,  9,    1891. 

*  Koch  und  Wolff hiigel,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1,  1882. 
^  Koch,  Gaffky  and  Loeffler,  ibid. 


66  BIOLOGY   AND   TECHNIQUE 

These  observers  were  able  to  show  that  the  spores  of  anthrax  were 
destroyed  by  boihng  water  at  100°  C.  in  from  one  to  twelve  minutes, 
whereas  dry  hot  air  was  efficient  only  after  three  hours'  exposure  to 
140°  C.  Extensive  confirmation  of  these  differences  has  been  brought 
by  many  workers.  An  explanation  of  the  phenomena  observed 
is  probably  to  be  found  in  the  changes  in  the  coagulability  of 
proteids  brought  about  in  them  by  the  abstraction  of  water. 
Lewith/  working  with  various  proteids,  found  that  these  sub- 
stances are  coagula+ed  by  heat  at  lower  temperatures  when  they 
contain  abundant  quantities  of  water,  than  when  water  has  been 
abstracted  from  them  On  the  basis  of  actual  experiment  with  egg 
albumin  he  obtained  the  following  results,^  which  illustrate  the  point 
in  question: 


Egg  albumin  in  dilute  aqueous  solution,  coagulated  at  56°  C. 
"       .  "         with  25  per  cent  water,  "  "  74-80°  C. 

u  u  u      jg    a        u  u  u  u  80-90°  C. 

«  it  li         o     a         a  ti  tc  {(    14K0  ri 


Absolutely  anhydrous  albumin,  according  to  Haas,^  may  be  heated 
to  170°  C.  without  coagulation.  It  is  thus  clear  that  bacteria  exposed 
to  hot  air  may  be  considerably  dehydrated  before  the  temperature  rises 
sufficiently  to  cause  death  by  coagulation,  complete  dehydration  neces- 
sitating their  destruction  possibly  by  actual  burning.    . 

Bacteria  exposed  to  moist  air  or  steam,  on  the  other  hand,  may  ab- 
sorb water  and  become  proportionately  more  coagulable. 

The  same  principle,  as  Lewith  points  out,  probably  explains  the  great 
resistance  to  heat  observed  in  the  case  of  the  highly  concentrated  pro- 
toplasm of  spores. 

Apart  from  the  actually  greater  efficiency  of  moist  heat  when  com- 
pared with  dry  heat  of  an  equal  temperature,  an  advantage  of  great 
practical  significance  possessed  by  moist  heat  lies  in  its  greater  powers  of 
penetration.  An  experiment  carried  out  by  Koch  and  his  associates 
illustrates  this  point  clearly.  Small  packages  of  garden  soil  were  sur- 
rounded by  varying  thicknesses  of  linen  with  thermometers  so  placed 
that  the  temperature  under  a  definite  number  of  layers  could  be  deter- 


1  Lewith,  Arch.  f.  exp.  Path.  u.  Pharm.,  xxvi,  1890. 

^Lewith,  loc.  cit.,  p.  351. 

8  Haas,  Prag.  med.  Woch.,  34-36,  1876, 


THE   DESTRUCTION   OF    BACTERIA 


67 


mined.    Exposures  to  hot  air  and  to  steam  were  then  made  for  com- 
parison, and  the  results  were  as  tabulated:* 


Tempera- 
tures. 

Time  of 
Application. 

Temperatures  Reached  within 
Thicknesses  of  Linen. 

Twenty 
Thicknesses. 

Forty 
Thicknesses. 

One  Hundred 
Thicknesses. 

Hot  air 

130-140°  C. 

4  hours. 

86° 

72° 

Below  70° 

Incomplete 
steriliza- 
tion. 

Steam 

90-105.3° 

3  hours. 

101° 

101° 

101.5° 

Complete 
steriliza- 
tion. 

This  great  penetrating  power  of  steam  is  due  presumably  to  its  com- 
paratively low  specific  gravity  which  enables  it  to  displace  air  from  the 
interior  of  porous  materials,  and  also  to  the  fact  that  as  the  steam  comes 
in  contact  with  the  objects  to  be  disinfected  a  condensation  takes  place 
with  the  consequent  liberation  of  he'at.  When  a  vapor  passes  into  the 
liquid  state  it  gives  out  a  definite  amount  of  heat,  which  in  the  case  of 
water  vapor,  at  100°  C,  amounts  to  about  537  calories.  This  brings 
about  a  rapid  heating  of  the  object  in  question.  Following  this 
process  the  further  heating  takes  place  by  conduction,  and  it  is,  of 
course,  well  known  that  steam  is  a  much  better  heat  conductor  than  air.^ 

Moist  heat  may  be  applied  as  boiling  water,  in  which,  of  course, 
the  temperature  varies  little  from  100°  C,  or  as  steam.  Steam  may  be 
used  as  live,  flowing  steam,  without  pressure,  the  temperature  of  which  is 
more  or  less  constant  at  100°  C,  or  still  higher  efficiency  may  be  attained 
by  the  use  of  steam  under  pressure,  in  which,  of  course,  temperatures 
far  Exceeding  100°  C.  may  be  produced,  according  to  the  amount  of 
pressure  which  is  used. 

The  spores  of  certain  bacteria  of  the  soil  which  can  not  be  killed  m 
live  steam  in  less  than  several  hours  may  be  destroyed  in  a  few  minutes, 
or  even  instantaneously,  in  compressed  steam  at  temperatures  ranging 
from  120°  to  140°  C." 

In  all  methods  of  steam  sterilization,  it  is  of  great  practical  impor- 


1  Koch,  Gaffky  und  Loeffler,  loc.  cit.,  p.  339. 

2  Gruber,  Cent.  f.  Bakt.,  iii,  1888. 

3  Christen,  Ref.  Cent.  f.  Bakt.,  V.  xiii,  1893. 


68  BIOLOGY   AND    TECHNIQUE 

V 

tance,  as  v.  Esmarch  ^  has  pointed  out,  that  the  steam  shall  be  saturated, 
that  is,  shall  contain  as  much  vaporized  water  as  its  temperature  per- 
mits. Unsaturated,  or  so-called  "  super-heated  steam  "  is  formed  when 
heat  is  applied  to  steam,  either  by  passage  through  heated  piping  or  over 
heated  metal  plates.  In  such  cases  the  temperature  of  the  steam  is 
raised,  but  no  further  water-vapor  being  supplied,  the  steam  exerts 
less  pressure  and  contains  less  water  in  proportion  to  its  volume  than 
saturated  steam  of  an  equal  temperature.  The  super-heated  steam, 
therefore,  is  heated  considerably  over  its  condensation  temperature  and 
becomes  literally  dried.  In  consequence,  its  action  is  more  comparable 
to  hot  air  than  to  saturated  steam,  and  up  to  a  certain  temperature  its 
disinfecting  power  is  actually  less  than  that  of  live  steam  at  100°  C. 
V.  Esmarch,  who  has  made  a  thorough  study  of  these  conditions,  con- 
cludes that  up  to  125°  C,  the  eflficiency  of  superheated  steam  is  lower 
than  that  of  live  steam  at  100°  C.  Above  this  temperature,  of  course, 
it  is  again  active  as  in  the  case  of  ordinary  dry  heat. 

Practical  Methods  of  Heat  Sterilization. — Burning. — ^For  ob- 
jects without  value,  actual  burning  in  a  furnace  is  a  certain  and  easily 
applicable  method  of  sterilization.  Flaming,  by  passage  through  a 
Bunsen  or  an  alcohol  flame,  is  the  method  in  use  for  the  sterilization  of 
platinum  needles,  coverslips,  or  other  small  objects  which  are  used  in 
handling  bacteria  in  the  laboratory. 

Hot  air  sterilization  is  carried  out  in  the  so-called  "hot  air  chambers,'^ 
simple  devices  of  varied  construction.  The  apparatus  most  commonly 
used  (Fig.  8)  consists  of  a  sheet-iron,  double-walled  chamber,  the 
joints  of  which,  instead  of  being  soldered,  are  closed  by  rivets.  The  inner 
case  of  this  chamber  is  entirely  closed  except  for  an  opening  in  the  top 
through  which  a  thermometer  may  be  introduced,  while  the  outer  has  a 
large  opening  at  the  bottom  and  two  smaller  ones  at  the  top.  A  gas- 
burner  is  adjusted  under  this  so  as  to  play  directly  upon  the  bottom  of 
the  inner  case.  A  thermometer  is  fitted  in  the  top  in  such  a  way  that  it 
penetrates  into  the  inner  chamber.  The  air  in  the  chamber  is  heated 
directly  by  the  flame  and  by  the  hot  air,  which,  rising  from  the  flame, 
courses  upward  within  the  jacket  between  the  two  cases  and  escapes  at 
the  top.  To  insure  absolute  sterilization  of  objects  in  such  a  chamber, 
the  temperature  should  be  kept  between  150°  and  160°  C.  for  at  least  an 
hour.  In  sterilizing  combustible  articles  in  such  a  chamber,  it  should  be 
remembered  that  cotton  is  browned  at  a  temperature  of  200°  C.  and 

1  V.  Esmarch,  Zeit.  f.  Hyg.,  iv,  1888. 


THE   DESTRUCTION   OF   BACTERIA 


69 


over.  This  method  is  used  in  laboratories  for  the  sterilization  of  Petri 
dishes,  flasks,  test  tubes,  and  pipettes,  and  for  articles  which  may  be  in- 
jured by  moisture.  Both  heating  and  subsequent  cooling  should  be  done 
gradually  to  avoid  cracking  of  the  glassware. 

Moist  /^eaf.— Instruments,  syringes,  and  other  suitable  objects  may 
be  sterilized  by  boiling  in  water.  Boiling  for  about  five  minutes  is  amply 
sufficient  to  destroy  the  vegetative  forms  of  all  bacteria.  For  the  de- 
struction of  spores,  boiling  for  one  or  two  hours  is  usually  sufficient, 
though  the  spores  of  certain  saprophytes  of  the  soil  have  been  found 


Fi©.  8. — Hot  Air  Sterilizer. 

occasionally  to  withstand  moist  heat  at  a  temperature  of  100°  C.  for 
as  long  as  sixteen  hours.  ^  The  addition  of  1  per  cent  of  sodium  car- 
bonate to  boiling  water  hastens  the  destruction  of  spores  and  prevents 
the  rusting  of  metal  objects  sterilized  in  this  way.  The  addition  of  car- 
bolic acid  to  boiling  water  in  from  2  to  5  per  cent  usually  insures  the 
destruction  of  anthrax  spores,  at  least,  within  ten  to  fifteen  minutes. 

Exposure  to  live  steam  is  probably  the  most  practical  of  the  methods 
of  heat  steriGzation.  It  may  be  carried  out  by  simple  makeshifts  of 
the  kitchen,  such  as  the  use  of  potato-steamers  or  of  wash-boilers.    For 


1  Christen,  loc.  cit. 


70 


BIOLOGY  AND  TECHNIQUE 


laboratory  purposes,  the  original  steaming  device  introduced  by  Koch 
has  been  almost  completely  displaced  by  devices  constructed  on  the 
plan  of  the  so-called  "  Arnold  "  sterilizer  (Fig.  9) .  In  such  an  appara- 
tus, water  is  poured  into  the  reservoir  A'  and  flows  from  there  into 
the  shallow  receptacle  B,  formed  by  the  double  bottom.  The  flame 
underneath  rapidly  vaporizes  the  thin  layer  of  water  contained  in  B, 
and  the  steam  rises  rapidly,  coursing  through  the  main  chamber  C. 
Steam  which  escapes  through  the  joints  of  the  lid  of  this  chamber  is 
condensed  under  the  hood  and  drops  back  into  the  reservoir.    Exposure 

to  steam  in  Tsuch  an  apparatus  for  fifteen 
to  thirty  minutes  insures  the  death  of 
the  vegetative  forms  of  bacteria. 

In  the  sterilization  of  media  by  such 
a  device,  the  method  of  fractional  sterili- 
zation at  100°  C.  is  employed.  The  prin- 
ciple of  this  method  depends  upon 
repeated  exposure  of  the  media  for  fif- 
teen minutes  to  one-half  hour  on  three 
succeeding  days.  By  the  first  exposure 
all  vegetative  forms  are  destroyed.  The 
media  may  then  be  left  at  room  tem- 
perature, or  at  incubator  temperature 
(37.5°  C.)  until  the  following  day,  when 
any  spores  which  may  be  present  will 
have. developed  into  the  vegetative  stage. 
These  are  then  killed  by  the  second  ex- 
posure. A  repetition  of  this  procedure 
on  a  third  day  insures  sterility.  It  must 
always  be  remembered,  however,  that 
this  method  is  applicable  only  in  cases 
in  which  the  substance  to  be  sterilized  is  a  favorable  medium  for 
bacterial  growth  in  which  it  is  likely  that  spores  will  develop  into  vege- 
tative forms. 

Exceptionally  the  method  may  fail  even  in  favorable  media  when 
anaerobic  spore-forming  bacteria  are  present.  Thus,  it  has  been  ob- 
served that  anaerobic  spores,  failing  to  develop  under  the  aerobic  con- 
ditions prevailing  during  the  intervals  of  fractional  steriWzation,  have 
developed  after  inoculation  of  the  media  with  other  bacteria,  when  sym- 
biosis had  made  their  growth  possible.  Tetanus  bacilli  have,  in  this  way, 
occurred  in  cultures  of  diphtheria  bacilli  employed  for  toxin  production. 


Fig.  9. — Arnold  Sterilizer, 


THE   DESTRUCTION   OF   BACTERIA  71 

In  noting  the  time  of  an  exposure  in  an  Arnold  sterilizer,  it  is 
important  to  time  the  process  from  the  time  when  the  temperature 
has  reached  100°  C.  and  not  from  the  time  of  Hghting  the  flame. 

The  principle  of  fractional  sterilization  at  low  temperatures  is  ap- 
plied also  to  the  sterilization  of  substances  which  can  not  be  sub- 
jected to  temperatures  as  high  as  100°  C.  This  is  especially  the  case 
in  the  sterilization  of  media  containing  albuminous  materials,  when 
coagulation  is  to  be  avoided,  or  when  both  coagulation  of  the  medium 
and  sterilization  are  desired. 

In  such  cases  fractional  sterilization  may  be  practiced  in  simply  con- 
structed sterilizers,  such  as  a  Koch  inspissator  or,  in  the  case  of  fluids, 
such  as  blood  serum,  by  immersion  in  a  water-bath  at  a  temperature 


Fig.  10.— Low  Temperature  Sterilizer  (Inspissator). 

varying  above  55°  C,  according  to  circumstances.  Exposures  at  such 
low  temperatures  may  be  repeated  on  five  or  six  consecutive  days,  usu- 
ally for  an  hour  each  day. 

The  use  of  steam  under  pressure  is  the  most  powerful  method  of  heat- 
disinfection  which  we  possess.  It  is  applicable  to  the  sterilization  of 
fomites,  clothing,  or  any  objects  of  a  size  suitable  to  be  contained  in  the 
apparatus  at  hand,  and  which  are  not  injured  by  moisture.  In  labora- 
tories this  method  is  employed  for  the  sterilization  of  infected  appa- 
ratus, such  as  flasks,  test  tubes,  Petri  plates,  etc.,  containing  cultures. 
The  device  most  commonly  used  in  laboratories  is  the  so-called  auto- 
clave, of  which  a  variety  of  models  may  be  obtained,  both  stationary 
and  portable.    The  pruiciple  governing  the  construction  of  all  of  these 


72 


BIOLOGY    AND    TECHNIQUE 


is  the  same.  The  apparatus  usually  consists  of  a  gun-metal  cylinder 
supplied  with  a  lid,  which  can  be  tightly  closed  by  screws  or  nuts, 
and  supplied  with  a  thermometer,  a  safety-valve,  and  a  steam  pressure 
gauge.  In  the  simpler  autoclaves,  water  may  be  directly  filled  into 
the  lower  part  of  the  cylinder,  and  the  objects  to  be  sterilized  supported 

upon  a  perforated  diaphragm.  In  this 
case  the  heat  is  directly  applied  by  means 
of  a  gas  flame.  In  the  more  elaborate 
stationary  devices,  steam  may  be  let  in 
by  piping  it  from  the  regular  supply  used 
for  heating  purposes.  Exposure  to  steam 
under  fifteen  pounds  pressure  (fifteen  in 
addition  to  the  usual  atmospheric  press- 
ure of  fifteen  pounds  to  the  square  inch) 
for  fifteen  to  twenty  minutes,  is  sufficient 
to  kill  all  forms  of  bacterial  life,  including 
spores. 

In  applying  autoclave  sterilization 
practically,  attention  must  be  paid  to 
certain  technical  details,  neglect  of  which 
would  result  in  failure  of  sterilization.  It 
is  necessary  always  to  permit  all  air  to 
escape  from  the  autoclave  before  closing 
the  vent.  If  this  is  not  done,  a  poorly 
conducting  air-jacket  may  be  left  about 
the  objects  to  be  sterilized,  and  these 
may  not  be  heated  to  the  temperature 
indicated  by  the  pressure.  It  is  also  nec- 
essary to  allow  the  reduction  of  pressure, 
after  sterilization,  to  take  place  slowly. 
Any  sudden  relief  of  pressure,  such  as 
would  be  produced  by  opening  the  air- 
vent  while  the  pressure  gauge  is  still  above  zero,  will  usually  result  in 
a  sudden  ebullition  of  fluid  and  a  removal  of  stoppers  from  flasks. 

The  temperature  attained  by  the  application  of  various  degrees  of 
pressure  is  expressed  in  the  following  table : 


Fig.  11. — Autoclave. 


Lbs.  Pressure  Temperature 

1  102.3° 

2 104.2 

3 105.7 

4 X07.3 


Lbs.  Pressure  Temperature 

5  108.8° 

6 110.3 

7 111.7 

8 113 


THE    riiSTRUCTION    OF   BACTERIA 


73 


Lbs.  Pressure  Temperature 

9  114.3° 

10 115.6 

11 116.8 


12 
13 
14 
15 
16 


118 

119.1 

120.2 

121.3 

122.4 


Lbs.  Pressure  Temperature 

17 123.3° 

18 124.3 

20 126.2 

22 128.1 

24 129.3 

26 :....  131.5 

28 133.1 

30 134.6 


CHEMICAL    AGENTS    INJURIOUS    TO    BACTERIA 


Since  the  time  of  Koch's  ^  fundamental  researches  upon  chemical 
disinfectants,  the  known  number  of  these  substances  has  been  enor- 
mously increased,  and  now  embraces  chemical  agents  of  the  most  varied 
constitution.  It  is  thus  manifestly  impossible  to  refer  the  injurious  in- 
fluence which  these  substances  exert  upon  bacteria  to  any  uniform  law  of 
action.  The  efficiency  of  a  disinfecting  agent,  furthermore,  is  not  alone 
dependent  upon  the  nature  and  concentrations  of  the  substance  itself,  but 
depends  complexly  upon  the  nature  of  the  solvent  in  which  it  is  employed, 
the  temperature  prevailing  during  its  application,  the  numbers  and  bio- 
logical characteristics  of  the  bacteria  in  question,  and  the  time  of  ex- 
posure. All  these  factors,  therefore,  must  be  considered  in  testing  the 
efficiency  of  any  given  disinfectant.  While  it  is  true,  furthermore, 
that  all  substances  which  in  a  given  concentration  exert  bactericidal  or 
disinfecting  action  upon  a  microorganism,  will  in  greater  dilution  act 
•antiseptic ally  or  inhibitively,  no  definite  rules  of  proportion  exist  be- 
tween the  two  values,  which  in  each  case  must  be  determined  by  experi- 
ment. 

Disinfectants  Used  in  Solution. — The  actual  processes  which  take  place 
in  the  injury  of  bacteria  by  disinfectants  are  to  a  large  extent  unknown. 
In  the  case  of  strong  acids,  or  strongly  oxidizing  substances,  there  may 
be  destruction  of  the  bacterial  body  as  a  whole  by  rapid  oxidation. 
Other  substances  may  act  by  coagulation  of  the  bacterial  protoplasm; 
others  again  by  diffusion  through  the  cell  membrane  are  able  to  enter  into 
chemical  combination  with  the  protoplasm  and  exert  a  toxic  action. 
Again,  in  other  cases,  a' difference  in  tonicity  between  cell  protoplasm 
and  disinfectant  may  tend  to  withdrawal  of  water  from  the  bacterial 
cell  and  consequent  injury  of  the  microorganism. 

Among  the  inorganic  disinfectants  the  most  important  are  the  metallic 

1  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 


74  BIOLOGY   AND   TECHNIQUE 

salts,  acids,  and  bases,  the  halogens  and  their  derivatives,  and  certain 
oxidizing  agents  like  peroxide  of  hydrogen  and  permanganate  of  potas- 
sium. 

It  has  been  shown  by  Scheuerlen  and  Spiro,^  Kronig  and  Paul,^  and 
others,  that  in  the  case  of  the  salts,  acids,  and  bases,  there  is  a  distinct 
and  demonstrable  relationship  between  the  disinfecting  power  of  these 
substances  and  their  dissociation  in  solution. 

According  to  the  theory  of  electrolytic  dissociation,  when  bodies  of 
this  class  go  into  solution  they  are  broken  up  or  dissociated  into  an 
electro-positive  and  an  electro-negative  ion.  Thus,  metallic  salts  are 
broken  up  into  the  kation,  or  positive  metal,  and  into  the  anion,  or 
negative  acid  radicle  (AgNOg  =  Ag,  +  ion  and  NO3,  —  ion).  In  the 
case  of  the  acids,  ionization  takes  place  into  the  hydrogen  ions  and  the 
acid  radicles,  while  in  the  case  of  the  bases  the  dissociation  occurs  into 
the  metal,  on  the  one  hand,  and  the  OH  group  on  the  other.  The  de- 
gree of  dissociation  taking  place  depends  upon  the  nature  of  the  sub- 
stance in  solution,  its  concentration,  and  the  nature  of  the  solvent. 
Thus,  in  any  such  solution  there  appear  three  substances,  the  undis- 
sociated  compound  as  such,  its  electro-negative  ion,  and  its  electro- 
positive ion,  their  relative  concentrations  depending  upon  an  interrela- 
tionship calculable  by  definite  laws.  It  goes  without  saying,  therefore, 
that  any  chemical  or  physical  reaction,  taken  part  in  by  such  a  solution, 
may  be  participated  in,  not  only  by  the  dissolved  undissociated  residue 
as  a  whole,  but  by  its  separate  ions  individually  as  well.  In  the  case  of 
many  disinfectants,  the  writers  referred  to  above  have  been  able  to 
demonstrate  a  relationship  between  the  degree  of  dissociation  and  the 
bactericidal  powers.  According  to  Kronig  and  Paul,  double  metallic 
salts,  in  which  the  metal  is  a  constituent  of  a  complex  ion  and  in  which 
the  concentration  of  the  dissociated  metal-ions  is  consequently  low, 
have  very  little  disinfecting  power.  Thus  potassium-silver-cyanide, 
which  is  a  comparatively  weak  disinfectant,  dissociates  into  the  kation  K 
and  the  complex  anion  Ag  (GN)  2,  this  latter  further  dissociating  to  a  very 
slight  degree  only.  The  same  writers  conclude  that  the  bactericidal 
action  of  mercuric  chloride  and  of  halogen  combinations  with  metals  is 
directly  proportionate  to  the  degree  of  dissociation.  This  considera- 
tion, moreover,  explains  why  aqueous  solutions  of  such  substances  are 
more  active  than  are  solutions  in  the  alcohols  or  in  ether,  since  it  is  well 


1  Scheuerlen  und  Sjdro,  Munch,  med.  Woch.,  44, 1897. 

2  Kronig  und  Paul,  Zeit.  f.  Hyg.,  xxv,  1897. 


THE  DESTRUCTION  OF  BACTERIA  75 

known  that  metallic  salts  are  ionized  in  these  substances  to  a  much 
sUghter  degree  than  they  are  in  water. ^ 

On  the  other  hand,  the  addition  of  moderate  quantities  of  ethyl 
and  methyl  alcohol  or  acetones  to  aqueous  solutions  of  silver  nitrate  or 
mercuric  chloride,  definitely  increases  the  disinfecting  action  of  such 
solutions.  In  the  case  of  mercuric  chloride,  Kronig  and  Paul  obtained 
the-  most  powerful  effects  in  solutions  to  which  alcohol  had  been  added 
in  a  concentration  of  25  per  cent.  For  this  empirical  fact  a  satisfactory 
explanation  has  not  yet  been  found.  Kronig  and  Paul  suggest  that  low 
percentages  of  alcohol  may  facilitate  the  penetration  of  the  disinfectant 
through  the  cell  membrane  and  thus  increase  its  efficiency,  while  high 
percentages  of  alcohol  have  the  opposite  effect,  by  decreasing  the  degree 
of  dissociation.  *In  this  connection  it  has  been  suggested,  however, 
that  absolute  and  strong  alcohols  possibly  act  as  desiccating  agents, 
thus  actually  rendering  the  bacteria  dry  and  less  susceptible  to  dele- 
terious chemical  influences. 

In  the  case  of  acids  and  bases  the  same  authors  have  determined 
that  the  powers  of  disinfection  of  these  substances  are  again  directly 
proportionate  to  the  degree  of  their  dissociation:  that  is,  to  the  concen- 
tration of  the  hydrogen  or  hydroxyl  ions,  respectively.  The  hydrogen 
ions  are  more  powerfully  active  than  the  hydroxyl  ions  in  equal  con- 
centration; acids,  therefore,  are  more  efficient  disinfectants  than  bases. 

A  fact  which  appears  to  strengthen  the  opinion  as  to  the  relationship 
between  bactericidal  powers  and  dissociation,  is  that  brought  forward 
by  Scheuerlen  and  Spiro,  that  the  addition  of  NaCl  to  bichloride  of 
mercury  solutions  reduces  the  disinfecting  power  of  such  solutions,  in- 
asmuch as  it  diminishes  the  concentration  of  free  ions.  In  practice, 
however,  NaCl  or  NH4CI  is  added  to  bichloride  of  mercury  solutions, 
since  these  substances  aid  in  holding  in  solution  mercury  compounds 
formed  in  the  presence  of  alkaUne  albuminous  material,  blood  serum, 
pus,  etc. 

In  regard  to  the  halogens,  Kronig  and  Paul  have  shown  that  the 
germicidal  power  of  this  class  of  elements  is  inversely  proportionate  to 
their  atomic  weights.  Thus,  chlorine  with  the  lowest  atomic  weight  is  the 
strongest  disinfectant  of  the  group.     Next,  and  almost  equal  to  this,  is 

1  Water  is  the  strongest  dissociant  known.  Methyl  alcohol  has  about  one-half  to 
two-thirds  the  dissociating  power  of  water  (Zelinsky,  Zeit.  f.  physiol. -Chemie,  xx, 
1896).  Ethyl  alcohol  allows  dissociation  much  less  than  methyl  alcohol;  ammonia 
allows  dissociation  to  about  one-third  to  one-fourth  the  extent  of  water.  See  Jones, 
"Elements  of  Physical  Chemistry,"  p.  371.     Macmillan,  New  York,  1902. 


76  BIOLOGY  AND  TECHNIQUE 

bromine.    Iodine  with  a  much  heavier  atomic  weight  than  either  of  the 
former  is  distinctly  less  bactericidal. 

Chloride  op  Lime. — Of  the  halogen  compounds  used  in  practice, 
the  most  important  is  chloride  of  lime  or  bleaching  powder.  As  to  the 
composition  of  this  substance,  there  is  some  difference  of  opinion.  It 
was  formerly  believed  to  be  a  mixture  of  calcium  hypochlorite, 
CaCClOg),  and  of  calcium  chloride,  CaClg.  The  fact  that  the  substance 
is  not  deliquescent,  however,  speaks  against  the  presence  of  calcium 
chloride  as  such,  and  it  is  probable  that  it  consists  of  a  single  com- 
pound with  the  formula  CaOClg.  The  action  of  acids  or  even  of 
atmospheric  COo  upon  this  substance  results  in  the  liberation  of 
chlorine.    For  instance, 

CaCCl^O)  +  2HC1  =  CaClg  +  2HC10. 
2HC10       +  2HC1  =  2H2  +  2CI2. 

Bleaching  powder  is  readily  soluble  in  about  twenty  parts  of  water. 
According  to  Nissen,^  solutions  of  2  in  1,000  of  this  substance  destroy 
vegetative  forms  of  bacteria  in  five  to  ten  minutes.  Its  bactericidal 
action  depends  on  the  hypochlorous  acid  formed.  After  water  precipi- 
tation an  efficient  dosage  is  10  pounds  to  the  million  gallons. 

Terchloride  of  Iodine  (ICI3)  is  an  extremely  strong  disinfectant, 
being  efficient  for  vegetative  forms  in  solutions  of  0.1  per  cent  in  one 
minute  and  a  1  per  cent  solution  destroying  spores  within  a  few 
minutes.^ 

Painting  with  tincture  of  iodine  (10  per  cent)  is  a  simple  and 
reliable  method  of  sterilizing  the  skin.  It  is  now  used  in  many  clinics 
in  sterilizing  the  field  of  operation. 

Peroxide  of  Hydrogen  is  formed  by  the  action  of  dilute  sulphuric 
acid  upon  peroxide  of  barium.  It  readily  gives  up  oxygen  and  acts 
upon  bacteria  probably  by  virtue  of  the  liberation  of  nascent  oxygen. 
In  the  presence  of  organic  matter,  such  as  blood,  pus,  etc.,  associated 
with  bacteria,  II2O2  is  quickly  reduced  and  weakened.  It  is  important 
that  the  HgOg  come  in  immediate  contact  with  the  bacteria.  In  prac- 
tice, therefore,  blood  and  pus  should  be  removed  from  wounds  when 
applying  the  H2Q2  or  a  large  excess  of  H2O2  should  be  used. 

Permanganate  of  Potassium,  acting  probably  in  the  same  way,  is 
a  powerful  germicide.  It  also  is  readily  reduced  by  many  organic  sub- 
stances often  associated  with  bacteria,  being  rendered  weaker  thereby. 

1  Nissen,  Zeit.  f .  Hyg.,  viii,  1890.  2  y,  Behring,  Zeit.  f.  Hyg.,  ix,  1891. 


THE    DESTRUCTION   OF    BACTERIA  77 

Among  organic  disinfectants  those  of  most  practical  importance  are 
the  alcohols,  formaldehydes,  iodoform,  members  of  the  phenol  group 
and  its  derivatives,  carbolic  acid,  cresol,  lysol,  creolin,  salicylic  acid,  cer- 
tain ethereal  oils,  and,  more  recently  introduced,  organic  silver  salts 
such  as  protargol,  argyrol,  argonin,  and  others. 

The  alcohols  are  but  indifferent  disinfectants.  Koch  ^  in  1881 
found  that  anthrax  spores  remained  alive  for  as  long  as  four  months 
when  immersed  in  absolute  and  in  50  per  cent  ethyl  alcohol.  On  the 
other  hand,  while  absolute  alcohol  possesses  practically  no  germicidal 
powers,  possibly  because  of  the  forrnation  of  a  protecting  envelope  by 
the  coagulation  of  the  bacterial  ectoplasm,  or,  as  suggested  above,  by 
desiccation  due  to  the  abstraction  of  water,  dilute  alcohol  in  a  concen- 
tration of  about  50  per  cent  is  distinctly  germicidal,  destroying  the  vege- 
tative forms  of  bacteria  in  from  ten  to  fifteen  minutes  or  less.^ 
Attention  has  already  been  called  to  the  fact  that  moderate  ad- 
ditions of  alcohol  to  aqueous  solutions  of  mercuric  chloride  enhance 
the  germicidal  power  of  this  disinfectant.  Additions  of  ethyl  and 
methyl  alcohol  to  carbolic  acid  or  formaldehyde  solutions,  on  the 
other  hand,  progressively  decrease  the  bactericidal  activities  of  these 
substances.^ 

The  value  of  boiling  alcohol  for  the  destruction  of  spores — especially 
in  the  sterilization  of  catgut — has  been  investigated  by  Saul,'*  who 
found  that  boiling  in  absolute  ethyl,  methyl,  or  propyl  alcohol  is  prac- 
tically without  effect,  while  spores  are  destroyed  readily  in  boiling 
dilute  alcohol,  the  most  effectual  being  propyl  alcohol  of  a  concentra- 
tion of  from  10-40  per  cent. 

Iodoform  (CHIg)^  is  weakly  antiseptic  in  itself,  but  when  introduced 
intD  wounds  where  active  reducing  processes  are  taking  place — often 
as  the  result  of  bacterial  growth — iodine  is  liberated  from  it  and  active 
bactericidal  action  results. 

Carbolic  acid  (CgHgOH),  at  room  temperature,  consists  of  color- 
less crystals  which  become  completely  liquefied  by  the  addition  of  10 
per  cent  of  water.  In  contradistinction  to  most  inorganic  disinfectants, 
the  action  of  carbolic  acid  and  other  members  of  the  phenol  group  is 


1  Koch.  Arb.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 

2  Epstein,  Zeit.  f.  Hyg.,  xxiv,  1897. 

3  Kronig  und  Paul,  loc.  cit. 

*  Saul,  Archiv  f.  klin.  Chir.,  56,  1898. 

6  V.  Behring,  "  Bekaempfung  d.  Infektions-Krankh.,"  Leipzig,  1894. 


78  BIOLOGY   AND   TECHNIQUE 

not  in  any  way  dependent  upon  dissociation.^  According  to  Beckmann  ^ 
and  others,  carbolic  acid  acts  as  a  molecule  and  not  by  individual  ions. 
The  proof  of  this  is  brought  out  by  the  fact  that  the  addition  of  NaCl 
to  carbolic  acid  solutions,  an  addition  which  would  tend  to  decrease 
the  concentration  of  free  ions,  markedly  increases  the  bactericidal 
powers  of  such  solutions.  On  the  other  hand,  as  stated  above,  addi- 
tions of  alcohol  progressively  diminish  the  efficiency  of  the  phenols. 

Other  members  of  this  group  of  disinfectants  are  ortho-,  meta-,  and 
PARACRESOL  (Ce5H4CH30H) ,  isomeric  compounds  differing  only  in  the 
position  of  the  OH  radicle.  Tricresol  is  a  mixture  of  these  three.  The 
cresols  are  relatively  more  powerfully  germicidal  than  is  carbolic  acid, 
but  are  less  soluble  in  water.  Lysol  is  a  substance  obtained  by  the 
solution  of  coal-tar  cresol  in  neutral  potassium-soap.  Dissolved  in 
water  it  forms  an  opalescent  easily  flowing  liquid.  According  to  Gru- 
ber,^  its  germicidal  action  is  slightly  greater  than  that  of  carbolic  acid. 
Creolin,  another  combination  of  the  cresols  with  potassic  soap,  forms 
with  water  a  turbid  emulsion,  v.  Behring  ^  expressed  the  relative 
germicidal  powers  of  carbolic  acid,  cresol,  and  creolin  for  vegetative 
forms  by  the  numbers  1:4   :   10,  in  the  order  named. 

Formaldehyde  (H-COH),  or  methyl  aldehyde,  is  a  gas  which  is 
easily  produced  by  the  incomplete  combustion  of  methyl  alcohol.  The 
methods  of  actually  generating  it  for  purposes  of  fumigation  will  be 
discussed  in  a  subsequent  paragraph.  In  aqueous  solution  this  substance 
forms  a  colorless  liquid  with  a  characteristic  acrid  odor,  and  in  this  form 
is  largely  used  as  a  preservative  for  animal  tissues  and  as  a  germicide. 
It  is  marketed  as  "formalin,"  which  is  an  aqueous  solution  containing 
from  35  to  40  per  cent  of  the  gas  and  which  exerts  distinctly  bactericidal 
action  on  vegetative  forms  in  further  dilutions  of  from  1  to  10  to  1  to 
20  (formaldehyde  gas  1  :  400  to  1  :  800) .  Anthrax  spores  are  killed 
in  35  per  cent  formaldehyde  in  ten  to  thirty  minutes.^  Unlike  the 
phenols,  the  addition  of  salt  to  formaldehyde  solutions  does  not  increase 
its  efficiency,  but  similar  to  them,  additions  of  ethyl  and  methyl  alcohol 
markedly  reduce  its  germicidal  powers. 

The  essential  oils  which  are  most  commonly  used  in  practice — 
largely  as  intestinal  antiseptics — are  those  of  cinnamon,  thyme,  eucalyp- 

^  Scheuerlen  und  Spiro,  Miinch.  med.  Woch.,  44,  1897. 
2  Beckmann,  Cent.  f.  Bakt.,  I,  xx,  1896. 
8  Gruber,  Cent.  f.  Bakt,  I.,  xi,  1892. 
*v.  Behring,  loc.  cit.,  p.  111. 
» Kronig  und  Paul,  loc.  cit. 


THE    DESTRUCTION    OF    BACTERIA  70 

tus,  and  peppermint.  Omeltschenko  ^  believes  that  the  employment  of 
these  oils  in  emulsions  is  illogical,  inasmuch  as  their  bactericidal  powers 
depend  upon  their  vaporization.  He  classifies  the  oils  in  decreasing 
order  of  their  efficiencj^  as  follows:  Oil  of  cinnamon,  prunol,  oil  of  thyme, 
oil  of  peppermint,  oil  of  camphor,  and  eucal3^ptol. 

Methods  of  Testing  the  Efficiency  of  Disinfectants.— The  efficiency  of 
any  given  disinfectant  depends,  as  we  have  seen,  upon  a  number  of 
factors,  any  one  of  which,  if  variable,  may  lead  to  considerable  differences 
in  the  end  result.  Thus,  as  far  as  the  bacteria  themselves  are  concerned, 
it  is  necessary  to  remember  that  not  only  do  separate  species  differ  in 
their  resistance  to  disinfectants,  but  that  different  strains  within  the 
same  species  may  show  such  variations  as  well.  This  fact  largely  ac- 
counts for  the  widely  varying  reports  made  by  different  investigators 
as  to  the  resistance  of  anthrax  spores,  and  depends  possibly  upon  tem- 
porary or  permanent  biological  differences  produced  in  bacteria  by  the 
conditions  of  their  previous  environment. 

The  numbers  of  bacteria  exposed  to  the  disinfectant,  furthermore, 
is  a  factor  which  should  be  kept  constant  in  comparative  tests.  The 
medium,  moreover,  in  which  bacteria  are  brought  into  contact  with  the 
disinfectant  is  a  matter  of  great  importance,  inasmuch  as  either  by 
entering  into  chemical  combination  with  the  disinfectant  it  m.ay  detract 
from  its  concentration  or  by  coagulation  it  may  form  a  purely  mechanical 
protection  for  the  microorganism.  Thus  bacteria  which  may  be  de- 
stroyed in  distilled  water  or  salt-solution  emulsion  with  comparative 
ease,  may  evince  an  apparently  higher  resistance  if  acted  upon  in 
the  presence  of  blood  serum,  mucus,  or  other  albuminous  substances. 
Temperature  influences  bactericidal  processes  in  that  most  chemical 
disinfectants  are  more  actively  bactericidal  at  higher  than  at  lower 
temperatures,  a  fact  due  most  likely  to  the  favorable  influence  of  tem- 
perature upon  all  chemical  reactions.^  As  far  as  merely  inhibitory  or 
antiseptic  values  are  concerned,  however,  the  temperature  least  favor- 
able for  the  reaction  of  the  antiseptic  is  that  which  represents  the  opti- 
mum growth  temperature  for  the  microorganism  in  question  and  the 
inhibitory  effects  of  any  substance  are  less  marked  at  this  point  than  at 
temperatures  above  or  below  it. 

The    important    influence   exerted   by  the    solvent   in    which   the 


1  Omeltschenko,  Cent.  f.  Bakt.,  I,  ix,  1891. 

'  V.  Behring,  "  Bekaempf.  der  Infektions-Krankh.,  Infektion  u.  Desinfection," 
Leipzig,  1894. 

7 


so 


BIOLOGY  AND  f ECHNIQtJE 


disinfectant  is  employed  has  already  been  discussed.  For  ordinary 
work  it  is  customary  to  express  absolute  and  comparative  antiseptic 
and  bactericidal  values  in  terms  of  percentages  based  upon  weight,  and 
this,  beyond  question,  is  both  simple  and  practical.  For  strictly  scien- 
tific comparisons,  however,  as  Kronig  and  Paul  ^  have  pointed  out,  it 
is  by  far  more  accurate  to  work  with  equimolecular  solutions. 

Rideal  and  Walker  ^  have  devised  a  method  of  testing  disinfectants, 
in  which  an  attempt  is  made  to  establish  a  standard  for  comparisons. 
They  choose,  as  the  standard,  carbolic  acid,  and  establish  what  they  call 
the  " carboHc-acid  coefficient."  This  coefficient  they  obtain  in  the  fol- 
lowing way:  the  particular  dilution  of  the  disinfectant  under  investiga- 
tion which  will  kill  in  a  given  time,  is  divided  by  the  strength  of  carbolic 
acid  which,  under  the  same  conditions,  will  kill  the  same  bacteria  in 
the  same  time.  We  quote  an  example  of  such  a  test,  given  by  Simpson 
and  Hewlett,^  comparing  formalin  and  carbolic  acid. 

BACILLUS  PESTIS. 


Dilution. 

Time  in  Minutes. 

Sample. 

2.5 

5 

7.5 

10 

12.5 

15 

Formalin -] 

Carbolic  acid -j 

lin    30 
lin    40 
1  in  100 
1  in  110 

growth 
growth 

growth 

growth 
growth 

growth 

growth 

...... 





In  the  above  table,  formalin  1  in  30  killed  in  the  same  time  as 
carbolic  acid  1  in  110.  Thus  the  carboUc-acid  coefficient  of  formalin 
in  this  test  =  ^%io  =  .27. 

The  Rideal- Walker  method  has  been  much  used  and  is  recommended 
by  many  workers.^ 

The  most  precise  method  of  standardizing  disinfectants  is  that  now 
in  use  in  the  U.  S.  Public  Health  Service.  It  is  a  modification  of  the 
Rideal- Walker  procedure  devised  by  Anderson  and  McClintic.^ 

Stock  5  per  cent  solutions  of  the  disinfectant  in  question  and  of  the 


1  Kronig  und  Paul,  loc.  cit 

2  Rideal  and  Walker,  Jour,  of  the  Sanitary  Ins.  London,  xxiv. 

3  Simpson  and  Hewlett,  Lancet,  ii,  1904. 
*  Sommerville,  Brit.  Med.  Jour.,  1904. 

^Anderson  and  McClintic,  Jour,  of  Inf.  Dis.,  1911,  viil,  1- 


THE  DESTRUCTION  OF  BACTERIA  81 

standard  (phenol)  are  first  prepared  and  a  series  of  accurate  dilutions 
made  with  distilled  water  using  graduated  pipettes.  (To  make  1 :  70  take 
4  c.c.  of  stock  and  10  c.c.  distilled  water;  1:80  =  4  c.c.  of  stock  +  12 
c.c.  distilled  water;  1:90  =  4  c.c.  stock  +  14  c.c.  distilled  water;  1:500 
=  2  c.c.  of  stock  +  48  c.c.  of  distilled  water.  Complete  dilution  tables 
are  given  in  their  original  article.)  The  series  should  include  dilutions 
strong  enough  to  kill  B.  typhosus  in  two  and  a  half  minutes  and  weak 
enough  to  fail  to  do  so  in  fifteen  minutes.  If  dilutions  greater  than  1- 
500  are  required,  a  second  1  per  cent  stock  solution  is  prepared.  They 
adopted  the  following  scale  for  their  tests :  Dilutions  up  to  1 :  70  should 
vary  from  the  next  in  the  series  by  a  difference  of  5  (i.e.,  5 parts  of  water). 

From  1:70      to  1:160    by  a  difference  of  10     ,0 
From  1 :  160    to  1 :  200    by  a  difference  of  20     ||^' 
From  1:200    to  1:400    by  a  difference  of  25      "'l' 
From  1 :  400    to  1 :  900    by  a  difference  of  50 
From  1 :  900    to  1 :  1800  by  a  difference  of  100 
From  1 :  1800  to  1 :  3200  by  a  difference  of  200 

and  so  on  if  higher  dilutions  are  necessary. 

Short  wide  test  tubes  1  inch  by  3  inches  are  used  in  making  the  test. 
These  are  placed  in  a  rack  in  a  water  bath  at  20°  C.  Five  c.c.  of  each 
dilution  are  measured  into  a  series  of  these  tubes  beginning  with  the 
strongest  specimen  and  rinsing  the  pipette  once  with  each  dilution 
before  the  5  c.c.  are  measured  out.  For  inoculation,  a  24-hour  broth 
culture  of  B.  typhosus  is  prepared  which  has  been  transferred  daily  for 
at  least  3  days.  Before  use  it  is  shaken  and  filtered  through  sterile 
filter  paper.  The  wide  test  tubes  containing  diluted  disinfectant  are 
inoculated  with  /lo  c.c.  of  this  culture  with  a  graduated  pipette.  The 
tip  of  the  pipette  is  held  against  the  side  of  the  tube  to  insure  accurate 
measurement  and  the  tube  immediately  shaken  to  mix  the  bacteria 
thoroughly  with  the  disinfectant.  Test  inoculations  are  made  from 
this  mixture  at  proper  intervals  into  tubes  containing  10  c.c.  of  standard 
extract  broth  of  +  1.5  acidity,  using  loops  4  mm.  in  diameter.  At  least 
four  such  loops  should  be  at  hand,  supported  on  a  rack  or  wooden  block 
so  that  a  fan-tail  Bunsen  burner  may  be  placed  under  each  wire  in  turn. 
Each  one  is  sterilized  after  a  plant  is  made  and  allowed  to  cool  while  the 
other  three  are  being  used  in  order. 

The  test  is  conducted  as  follows:  A  row  of  ten  wide  tubes  containing 
dilutions  of  the  antiseptic  is  placed  in  the  water  bath  at  20°  C.  and  time 
allowed  for  them  to  reach  the  temperature  of  the  bath.  They  are  then 
inoculated  in  order  at  intervals  of  exactly  15  seconds.    Fifteen  seconds 


82  BIOLOGY  AND  TECHNIQUE 

after  the  last  tube  has  been  inoculated  a  subculture  is  made  from  the 
first  tube  of  the  series  (i.e.,  23^  minutes  after  this  first  tube  was  inocu- 
lated) and  from  the  other  tubes  in  order  at  15-second  intervals.  Fifteen 
seconds  after  this  first  series  of  subcultures  is  completed  a  second  series 
of  subcultures  is  begun  which  will  give  the  result  of  a  5-minute  exposure  to 
the  antiseptic  and  the  subinoculations  continued  at  15-second  intervals 
until  all  dilutions  have  been  tested  for  fifteen  minutes.  If  the  strength 
of  the  antiseptic  is  known  approximately  subcultures  of  the  lower  dilu- 
tions for  the  longer  periods  may  be  omitted.  It  is  convenient  to  have 
an  assistant  at  hand  to  call  time  and  to  label  the  subcultures  as  soon  as 
made.     The^jbes  may,  however,  be  placed  in  order  in  suitable  racks 

DETP  .{m:  ATION  OF  THE  CARBOLIC-ACID  COEFFICIENT 
•!►  OF  A  DISINFECTANT. 

(Anderson  and  McClintic) 

Name "A" 

Temperature  of  Medication 20°  C. 

Culture  Used  B.  Typhosus 24-hr.,  Extract  Broth,  Filtered 

Proportion  of  Culture  and  Disinfectant 0.1  c.c.  +  5  c.c. 

Organic  Matter,  None;    Kind,  None;    Amount,  None. 

Subculture  Media Standard  Extract  Broth 

Reaction +  1.5 

Quantity  in  Each  Tube 10  c.c. 


Sample. 

Dilu- 
tion. 

Time  Culture  Exposed  to  Action 
of  Disinfectant  for  Minutes 

Phenol  Coefficient. 

2y2 

5 

7y2 

10 

12^ 

15 

Phenol 

1:80 
1:90 

+ 

— 

— 

80)375 

1:100 

+ 

+ 

+ 

— 

— 

— 

4.69 

1:110 

+ 

+ 

+ 

+ 

+ 

— 

110)650 

5.91 

Disinfectant  "A".. 

1:350 
1:375 

— 

— 

2)10.60 

1:400 

+ 

— 

— 

— 

5.30  = 

1:425 

+ 

+  ' 

— 

— 

— 

— 

coefficient 

1:450 

+ 

+ 

— 

— 

— 

1:500 

+ 

+ 

-r- 

— 

— 

— 

1:550 

+ 

+ 

+ 

— 

— 

— 

1:600 

+ 

+ 

+ 

+ 

— 

— 

1:650 

+ 

+ 

+ 

+ 

+ 

— 

1:700 

+ 

+ 

+ 

+ 

+ 

+ 

1:750 

+ 

+ 

+ 

+ 

+ 

+ 

THE  DESTRUCTION  OF  BACTERIA  83 

without  labelling.  The  subculture  tubes  are  incubated  for  48  hours  at 
37°  C.  and  those  in  which  growth  is  observed  are  recorded  positive. 

To  obtain  the  coefficient  the  weakest  dilution  of  the  unknown 
antiseptic  which  kills  in  23/^  minutes  is  divided  by  the  weakest  dilution 
of  phenol  which  kills  in  the  same  time.  The  same  is  done  for  the  weak- 
est strength  that  kills  in  15  minutes  and  an  average  is  taken.  The 
results  of  such  a  test  are  shown  in  the  table  on  page  82. 

As  only  the  23/^-minute  and  15-minute  intervals  are  used  in  deter- 
mining this  result  it  seems  unnecessary  to  make  plants  at  the  intervening 
periods  except  in  special  cases  where  more  detailed  information  is  desired. 

The  procedure  may  be  modified  by  adding  some  organic  substance 
such  as  killed  bacteria  to  the  diluted  antiseptic.  For  many  substances, 
e.g.,  bichloride  of  mercury,  the  antispetic  value  in  presence  of  organic 
matter  is  much  lower  than  in  watery  solution.  Anderson  and  McClintic 
insist  that  great  care  in  making  the  dilutions  and  rigid  adherence  to  a 
uniform  technique  are  necessary  to  obtain  consistent  results  in  such  tests. 

Determination  of  Antiseptic  Values. — The  antiseptic  or  in- 
hibitive  strength  of  a  chemical  substance,  sometimes  spoken  of 
as  the  ''coefficient  of  inhibition,"  is  determined  by  adding  to 
definite  quantities  of  a  given  culture  medium,  graded  percent- 
ages of  the  chemical  substance  which  is  being  investigated  and  plant- 
ing in  these  mixtures  equal  quantities  of  the  bacteria  in  question. 
The  medium  used  for  the  tests  may  be  nutrient  broth  or  melted  gelatin 
or  agar.  If  broth  is  used,  growth  is  estimated  by  turbidity  of  -the 
medium  and  by  morphological  examination;  if  the  agar  or  gelatin  is 
employed,  plates  may  be  poured  and  actual  growth  observed. 

Thus,  in  the  case  of  carbolic  acid,  a  5  or  10  per  cent  solution  is 
prepared  and  added  to  tubes  of  the  medium,  as  follows: 

Tube  1  contains  5%  carbolic  2  c.c.  +  broth  8      c.c.  =   1:  1,000  carbolic  acid. 

"      2         "  5            "  1  c.c.  +  broth  9      c.c.  =   1:200 

"      3         "  5            "  .5  c.c.  +  broth  9.5  c.c.  =  1:400          "           " 

"      4         "  5            "  7  c.c.  +  broth  9.8  c.c.  =   1:1,000      " 

"      5         "  5            "  .1  c.c.  +  broth  9.9  c.c.  =  1:5,000      "            " 

To  each  of  these  tubes  a  definite  quantity  of  the  bacteria  is  added 
either  by  means  of  a  standard  loopful  of  a  fresh  agar  culture,  or  better  by 
a  measured  volume  of  an  even  emulsion  in  sterile  salt  solution.  The 
inoculated  tubes  are  then  incubated  at  a  temperature  corresponding  to 
the  optimum  growth  temperature  for  the  microorganism  in  question. 
The  tubes  are  examined  for  growth  from  day  to  day.  From  tubes 
containing  higher  dilutions,  in  which  no  growth  is  visible,  transplants 


84 


BIOLOGY   AND    TECHNIQUE 


INHIBITION   STRENGTHS   OF   VARIOUS   ANTISEPTICS. 
Adapted  from  Flugge,  Leipzig,   1902. 


Acids 

Sulphuric 

Hydrochloric 


Sulphurous 
Arsenous . . , 
Boric    


Alkalies 
Potass,  hydrox . 


Ammon.  hydrox. 
Calcium  hydrox. 


Salts 

Copper  sulphate 
Ferric  sulphate 
Mercuric  chlorid . 
Silver  nitrate  .  . . 


Potass,  perman 

Halogens  and  Compounds 

Chlorin 

Bromin 

lodin 

Potass,  iodid 

Sodium  chlor 


Anthrax  Bacilli. 


1  :  3,000 
1 : 3.000 


1:800 
1:700 

1:700 


Organic  Compounds 

Ethyl  alcohol 

Acetic  and  oxalic  acids 
Carbolic  acid 


Benzoic  acid    

Salicylic  acid 

Formalin     (4%    formalde- 
hyde)  


Camphor 

Thymol   

Oil  mentha  pip    

Oil  of  terebinth 

Peroxide  of  hydrogen, 


1  :  100,000 
1  :  60,000 

1  : 1,000 

1  : 1,500 
1 : 1,500 
1  : 5,000 


Other  Bacteria. 


Choi.  spir.  1  :  6,000 
B.  diph.  1  :  3,000 
B.  mallei  1  :  700 
B.  typh.  1  :  500 
Choi.  spir.  1  :  1,000 


Putrefactive  Bac- 
teria in  Bouillon. 


B.  diphth.  1  :  600 
Choi.  spir.  1  :  400 
B.  typh.  1  :  400 
Choi.  spir.  1  :  500 
B.  typh.  1  :  500 
Choi.  spir.  1:1,100 
B.  typh.  1:1,100 


B.  typhosus  1  :  60,000 

Choi.  spir. 

B.  typhosus  1  :  50,000 


1:60 


1:12 


1:800 

1 : 1,000 
1 : 1,500 


1  : 1,000 
1  :  10,000 
1  :  3,000 
1 : 8,000 


B.  diph.  1  :  500 
B.  typh.  1  :  400 
Choi.  spir.  1  :  600 


Choi.  spir.  1  :  20,000 
Staphylo.    1  :  5,000 


1  : 6,000 

1:200 

1:100 


1  : 1,000 
1:90 
1  :  20,000 


1:500 

1  : 4,000 
1  : 2,000 
1:5,000 
1:7 


1:10 
1:400 


1 : 1,000 
1  : 3,500 
1  : 2,000 


THE  DESTRUCTION  OF  BACTERIA 


85 


BACTERICIDAL   STRENGTHS  OF   COMMON   DISINFECTANTS. 
Adapted  from  FlO^gge,  Leipzig,  1902. 


ACID8 

Sulphuric  ... 
Hydrochloric 
Sulphurous  . 


Sulphurous 
Boric 


Alkalies 
Potass,  hydrox. . 
Ammon.  hydrox. 
Calcium 


Salts 

Copper  sulphate 

Mercuric  chlor. 


Silver  nitrate  . . 
Potass,  permang. 
'''  Calc.  chlorid  " 


Halogens  and    Com 

POUNDS 

Chlorin 

Trichlorid  of  iodin . .  , 

Organic  Compounds 

Ethyl  alcohol   

Acetic  and  oxalic  acids 


Carbolic  acid 


Lysol 

Creolin 

SaHcylic  acid    

Formalin     (40%    for- 
maldehyde)      

Peroxide  of  hydrogen  . 


Strepto-  and 

Staphj^lo- 

cocci. 


5  Minutes. 


1:10 
1:10 


1:5 


1  :  10,000  to 

1,000 
1  :  1,000 
1  :200 


1  per  cent. 
1  :^200 


70%-15 
minutes 


1:60 
1  :300 


1  :  1,000 

1:10 
Cone. 


Anthrax  and  Tyfjhoid  Bacilli, 
Cholera   Spirillum. 


5  Minutes. 


1^100 
1:100 


1:300 
1:300 
1  : 1,000 


1  : 2,000 


1:500 


.1  per  cent. 
1  :  1,000 


70%-lOmins 


Cholera  1:200 
Typh.  1  :  50 
1:300 
1  :100 


1:20 
1  :200 


2-24  Hours. 


1  :  1,500 
1  :  1,500 
(Typhoid 
1:700) 
1:300   (Gas 
10  vol.  %) 
1:30 


1  :  10,000 
1  :  4,000 


1  : 2-300 


1.300 


1  : 3,000 


1  :  1,000 
1  :  500 


Anthrax  Spores. 


1  :  50  in  10  days 
1  :  50  in  10  days 


Cone.  sol.  incomplete 
disinfection 


1  :  20  (5  days) 
1:2,000  (26  hours) 


1  :  20  (1  day) 
1  :  20  (1  hour) 


2  per  cent  (in  1  hr.) 
1  :  1,000  (in  12  hrs.) 

Alcol.  50%  for  4 
months  without 
killing  spores. 

Koch.i 

1  :  20  (4-45  days) 
(at  40°  in  3  hrs.) 

(10%  in  5  hrs.) 


1  :  20  (in  6  hrs.) 
1  :  100  (in  1  hr.) 
3  :  100  (in  1  hr.) 


*  Koch.  Arb.  a.  d.  kais.  Gesundheitsamt,  1, 1881. 


86  BIOLOGY  AND  TECHNIQUE 

are  made  to  determine  the  presence  of  living  bacteria  and  to  distinguisli 
between  inhibition  or  antisepsis  and  bacterial  death  or  disinfection. 

The  determination  of  the  bactericidal  or  disinfectant  value  of  a 
chemical  substance  upon  spores  may  be  carried  out  by  a  variety  of 
methods.  Koch,^  using  anthrax  spores  as  the  indicator,  dried  the  spores 
upon  previously  sterilized  threads  of  silk.  These  were  exposed  to  the 
disinfectant  at  a  definite  temperature  for  varying  times,  the  disinfect- 
ant was  then  removed  by  washing  in  sterile  water,  and  the  threads 
planted  upon  gelatin  or  blood  serum  media  and  incubated.  A  serious 
objection  to  this  method  was  pointed  out  by  Geppert,^  who  maintains 
that  it  is  impossible  by  simple  washing  to  remove  completely  the  disin- 
fectant in  which  the  thread  has  been  soaked.  This  author  suggests  that, 
whenever  possible,  the  disinfectant,  at  the  end  of  the  time  of  exposure, 
should  be  removed  by  chemical  means.  In  the  case  of  bichloride  of  mer- 
cury Geppert  exposes  emulsions  of  the  bacteria  to  aqueous  solutions  of 
the  disinfectant,  and  at  the  end  of  exposure  precipitates  out  the  bichlor- 
ide of  mercury  with  ammonium  sulphide.  In  the  case  of  a  large  number 
of  disinfectants,  however,  this  is  not  possible,  and,  when  the  thread 
method  is  used,  removal  of  the  chemical  agent  by  washing  must  be 
practised.  Complete  removal  of  the  disinfectant  is  especially  desirable, 
since  spores  previously  exposed  to  these  substances  are  more  easily  in- 
hibited by  dilute  solutions  than  are  normal  spores.  The  spores  may  be 
dried  upon  the  end  of  a  glass  rod,  which,  after  exposure,  is  washed  in 
distilled  water  or  salt  solution  and  then  immersed  in  sterile  broth.^ 

A  simple  method  is  that  in  which  graded  percentages  of  the  disin- 
fectant are  added  to  the  menstruum,  blood,  blood  serum,  broth,  etc.,  in 
which  the  disinfectant  is  to  be  tested,  and  equal  quantities  of  bacteria 
thoroughly  emulsified  in  water  or  salt  solution  are  added.  Loopfuls  of 
these  mixtures  are  then  planted  from  time  to  time  in  agar  or  gelatin 
plates  upon  which  colony  counts  can  afterward  be  made. 

In  all  such  tests  it  is  important  to  remember  that  the  presence  of 
organic  fluids,  blood  serum,  mucus,  etc.,  considerably  alters  the  efficiency 
of  germicides,  and  whenever  practical  deductions  are  made,  experimental 
imitation  of  the  actual  conditions  should  be  attempted. 

Practical  Disinfection. — In  practical  disinfection  with  chemical 
agents,  the  disinfectant  must  be  chosen  to  a  certain  extent  in  accordance 
with  the  material  to  be  disinfected. 

1  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  1,  1881. 

2  Geppert,  Berl.  klin.  Woch.,  xxvi,  1889. 

» miL  Rep.  Am.  Pub.  Health  Assn.,  xxiv,  1898. 


THE  DESTRUCTION  OF  BACTERIA  ,  87 

Sputum  is  a  substance  extremely  difficult  to  disinfect  because  the 
bacteria  present  are  surrounded  by  dense  envelopes  of  mucus,  through 
which  disinfectants  do  not  easily  diffuse.  For  sputxmi  disinfection,  es- 
pecially tuberculous  sputum,  carboUc  acid — 5  per  cent  solution — or 
any  of  the  phenol  derivatives  in  similar  concentration,  may  be  used. 
Bichloride  of  mercury  is  of  very  little  use  in  sputum  disinfection  be- 
cause of  the  dense  protective  layers  of  albuminated  mercury  which  form 
about  the  microorganisms.  Sputum  should  always  be  received  into 
cups  containing  the  disinfectant,  and  contaminated  handkerchiefs 
should  be  soaked  in  the  solution. 

Feces  from  typhoid,  dysentery,  and  cholera  patients  should  be  steril- 
ized by  burning,  if  possible,  or  by  thoroughly  mixing  with  large  quan- 
tities of  boiling  water;  but  if  chemical  disinfectants  are  to  be  used,  five 
per  cent  carbolic  acid  or  dilute  formalin  are  convenient.  Milk  of  lime 
and  chloride  of  lime  are  useful,  though  somewhat  inconvenient.  Bichlo- 
ride of  mercury  is  of  little  value  in  this  case  for  the  same  reason 
that  it  is  valueless  in  sputum  disinfection.  In  all  cases  of  feces  dis- 
infection it  is  extremely  important  that  the  chemical  agent  should  be 
added  in  large  quantities  and  thoroughly  mixed  with  the  discharge. 

Ldnen,  napkins,  and  other  cloth  materials  which  have  come  into  con- 
tact with  patients  should  be  soaked  for  one  or  two  hours  in  one  per  cent 
formaldehyde,  five  per  cent  carbolic  acid,  or  1  : 5,000  or  1  :  10,000 
bichloride  of  mercury.  After  this,  the  material  may  be  taken  from  the 
sick-room  and  boiled.  It  is  extremely  important  that  cloth  material 
should  never  be  removed  from  the  sick-room  in  a  dry  state. 

Urine  may  be  easily  disinfected  by  the  addition  in  proper  con- 
centration of  any  of  the  disinfectants  named  above. 

The  methods  for  sterilization  of  surgical  instruments  and  the  prepara- 
tion of  the  skin  of  the  patient  for  operation  are  subject  to  so  many  local 
variations  that  it  is  hardly  within  the  scope  of  a  text-book  on  bacteriology 
to  mention  them.  Metal  instruments  are  usually  sterilized  by  boiling 
in  soda  solution  and  may  be  subsequently  immersed  in  five  per  cent  car- 
bolic acid  solution.  Catgut  may  be  sterilized  by  boiling  in  alcohol  or  by 
subjecting  it  to  temperatures  of  140°  C.  and  over,  for  several  hours  in 
oils  (albolin). 

The  disinfection  of  the  hands  is  also  a  matter  of  much  variation. 
Two  methods  frequently  quoted  are  those  of  Welch  and  of  Fiirbringer. 

In  Welch's  method  the  hands  are  brushed  with  green  soap  in  water 
as  hot  as  it  can  be  borne  for  at  least  five  minutes.  They  are  then  rinsed 
and  immersed  for  two  minutes  in  a  warm  saturated  solution  of  perman- 


88  ^  BIOLOGY  AND  TECHNIQUE 

ganate  of  potash  in  which  they  are  rubbed  with  a  sponge  or  sterile 
cotton.  They  are  then  transferred  to  a  saturated  solution  of  oxalic  acid, 
until  the  red  color  has  entirely  disappeared.  Following  this,  they  are 
rinsed  in  sterile  water  and  then  immersed  in  a  1  :  500  bichloride  of 
mercury  solution  for  one  to  two  minutes. 

According  to  Fiirbringer's  method,  the  finger  nails  are  carefully 
cleaned  with  an  orange-wood  stick  or  nail  file;  the  hands  are  then  thor- 
oughly brushed  with  a  nail  brush  in  green  soap  and  hot  water  for  five 
minutes.  Following  this  they  are  immersed  in  60  per  cent  alcohol  for 
one  minute,  then  in  3  per  cent  carbolic  acid  solution  for  one  minute; 
after  which  they  are  rinsed  in  sterile  water  and  dried. 

Rooms,  closets,  and  other  closed  spaces  which  are  contaminated,  must 
be  disinfected  largely  by  gaseous  disinfectants.  After  such  disinfection 
in  the  case  of  cellars,  privies,  and  other  places  where  feasible,  walls  and 
ceilings  should  be  whitewashed. 

Gaseous  Disinfectants  for  Purposes  of  Fumigation. — There  are  a 
large  number  of  gaseous  agents  which  are  harmful  to  bacteria.  Only  a 
few,  however,  are  of  sufficient  bactericidal  strength  to  be  of  practical 
importance. 

Oxygen,  especially  in  the  nascent  state,  may  exert  distinct  bacteri- 
cidal action  upon  some  bacteria.  That  strictly  anaerobic  strains  are 
inhibited  by  its  presence  has  already  been  mentioned. 

Ozone  was  shown  by  Ransome  and  Fullerton  ^  to  exert  considerable 
germicidal  power  when  passed  through  a  liquid  medium  in  which  bac- 
teria were  suspended,  but  was  absolutely  without  activity  when  em- 
ployed in  the  dry  state. 

Chlorine  because  of  its  powerful  germicidal  action  was  once  looked 
upon  with  favor,  but  has  been  found  quite  inadequate  from  a  practical 
point  of  view  because  of  its  injurious  action  upon  materials,  and  its 
irregularity  of  action.  Chlorine,  too,  is  but  weakly  efficient  unless  in 
the  presence  of  moisture.^ 

Sulphur  dioxide  or  sulphurous  an^ydrid  (SO2),  formerly  much  used 
for  room  disinfection,  is  no  longer  regarded  as  uniformly  efficient  for 
general  use.  The  gas  is  produced  by  burning  ordinary  roll  sulphur, 
conveniently  in  a  Dutch  oven.  To  be  at  all  effective,  water  should  be 
vaporized  at  the  same  time,  since  the  disinfectant  action  is  dependent 
upon  the  formation  of  sulphurous  acid.  The  concentration  of  the  gas 
should  be  at  least  8  per  cent  of  the  volume  of  air  in  the  room.    For  this 

^  Ransome  and  Fullerton,  Rep.  Public  Health,  July,  1901. 

*  Fischer  and  Proskauer,  Mitt.  a.  d.  kais.  Gesundheitsamt,  x,  11,  1882. 


THE  DESTRUCTION  OF  BACTERIA  89 

purpose  about  three  pounds  of  sulphur  should  be  burned  for  every 
thousand  cubic  feet  of  space.  It  should  be  allowed  to  act  for  not  less 
than  twenty-four  hours.  The  researches  both  of  Wolffhiigel^  and  of 
Koch  2  have  shown  that  the  gas  is  not  sufficient  for  the  destruction  of 
spores.  Park^  believes  that  sulphur  dioxid  used  in  quantities  of  four 
pounds  of  sulphur  to  1,000  cubic  feet  is  of  practical  value  for  fumigating 
purposes  in  cases  of  diphtheria  and  the  exanthemata.  Sulphur  dioxid 
fumigation  is  more  effective  than  formaldehyd  for  the  destruction  of 
insects — fleas,  lice  and  bedbugs — a  matter  of  importance  in  epidemics 
of  typhus  fever,  relapsing  fever,  plague,  etc. 

Of  all  known  gaseous  disinfectants  by  far  the  most  reliable  is  form- 
aldehyd. In  all  cases  where  formaldehyd  fumigation  is  intended, 
clothing,  bed-linen,  and  fabrics  should  be  spread  out,  cupboards  and 
drawers  freely  opened.  The  cracks  of  windows  and  doors  should  be 
hermetically  sealed  with  paper  strips  or  by  calking  with  cotton.  In  all 
cases  moisture  should  be  provided  for,  either  in  the  generating  appa- 
ratus or  by  a  separate  boiler. 

Direct  evaporation  of  formaldehyd  from  formalin  solutions  has  been 
the  principle  underlying  most  of  the  methods.  If  such  evaporation  is 
attempted  from  an  open  vessel,  however,  polymerization  of  formal- 
dehyd to  the  solid  trioxymethylene  occurs.  To  prevent  this,  Trillat* 
and  others  have  constructed  special  autoclaves  in  which  20  per  cent  of 
calcium  chloride  is  added  to  formalin  which  is  then  vaporized  under 
pressure. 

The  Trillat  autoclave,  as  well  as  others  constructed  on  the  same 
principle,  consists  of  a  strong  copper  chamber  of  a  capacity  of  about  a 
gallon,  fitted  with  a  cover  which  can  be  tightly  screwed  into  place. 
The  cover  is  perforated  by  an  outlet  vent,  a  pressure  gauge,  and  a 
thermometer.  The  whole  apparatus  is  adjusted  upon  a  stand  and  set 
over  a  kerosene  lamp.  Into  the  chamber  is  put  about  one-half  to  three- 
quarters  its  capacity  of  40  per  cent  formaldehyd  (commercial  formalin) 
containing  15-20  per  cent  calcium  chlorid.  The  solution  used  should 
be  free  from  methyl  alcohol,  since  this  leads  to  the  formation,  with 
formaldehyd,  of  methylal,  which  is  absolutely  without  germicidal 
action.    For  a  room  of  about  3,000  cubic  feet  Trillat  advises  the  con- 

^  Wolffhiigel,  Mitt.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 
2  Koch,  Mitt.  a.  d.  kais.  Gesundheitsamt,  i,  1881. 
'  Park,  "Pathogen.  Bact.,"  N.  Y.,  1908. 
*  Trillat,  Compt.  rend,  de  I'acad.  des  sc,  1896. 


90 


BIOLOGY  AND  TECHNIQUE 


tinuance  of  the  gas  flow  for  about  an  hour.  •  The  method  is  not  uni- 
formly reliable. 

A  method  which  has  found  much  favor  is  that  in  which  glycerin — 
usually  in  a  concentration  of  10  per  cent — is  added  to  formalin.  Ac- 
cording to  Schlossmann  ^  the  presence  of 
glycerin  hinders  polymerization.  An  appa- 
ratus in  which  this  mixture  is  conveniently 
utilized  is  that  of  Lentz  (see  Fig.  12).  For- 
malin with  10  per  cent  glycerin  is  placed 
in  the  copper  tank  and  heated  by  a  burner. 
This  apparatus  has  been  favorably  endorsed 
by  the  War  Department  of  the  United  States. 
The  so-called  Breslau  method  of  generat- 
ing formaldehyd  depends  upon  the  evapora- 
tion of  formaldehyd  from  dilute  solutions,  v. 
Brunn  ^  claims  that  where  formalin  in  30  to  40 
per  cent  strength  is  evaporated,  water  vapor 
is  generated  more  rapidly  than  formaldehyd 
is  liberated,  and  a  concentration  leading  to 
poljmaerization  occurs.  If,  however,  dilution 
is  carried  out  until  the  formaldehyd  in  the  solution  is  not  more  than 
8  per  cent,  the  generation  of  water  vapor  and  formaldehyd  take 
place  at  about  equal  speed  and  no  concentration  occurs.  Schlossmann  ^ 
furthermore  claims  that  polymerization  in  the  vaporized  formaldehyd 
does  not  occur  if  sufficient  water  vapor  is  present — a  principle  which 
may  also  contribute  to  the  efficiency  of  the  Breslau  method.  In  prac- 
tice, the  apparatus  devised  by  v.  Bruim  (Fig.  13)  consists  of  a  strong 
copper  kettle  of  about  34  cm.  diameter  by  7.5  cm.  height.  This  is 
completely  closed  except  for  two  openings  in  the  slightly  domed  top, 
one  of  which  is  the  exit  vent,  the  other,  laterally  placed,  is  for  pur- 
poses of  filling  and  is  closed  by  a  screw  stopper.  The  tank  is  filled 
with  a  solution  of  formalin  of  a  strength  of  from  8  to  10  per  cent  (com- 
mercial formalin  1:4).  The  apparatus  permits  the  evaporation  of 
large  quantities  of  fluid  in  a  short  time  (3  liters  in  one  hour).  When 
the  lamp  is  left  in  a  closed  room  care  should  be  taken  to  fill  it  with 
a  quantity  of  alcohol  proportionate  to  the  amount  of  fluid  to  be  evap- 
orated.    This,  according  to  v.  Brunn,  is  about  one-quarter  of  the 


Fig.  12. — Lentz  Formalin 
Apparatus. 


*  Schlossmann,  Munch,  med.  Woch.,  45,  1898. 

*  V.  Brunn,  Zeit.  f.  Hyg.,  xxx,  1899. 


THE  DESTRUCTION  OF  BACTERIA 


91 


,-<sS==^^^^^^ 


n 


Tj—^ ^r^ 

Fig.  13. — Breslau  Formaldehyd  Gen- 
erator AND  Section  of  Same.  (After  v. 
Brunn.)     a,  Inlet;  6,  Exit  vent. 


volume  of  formalin  solution  used.  By  using  1.5  liters  of  8  per  cent 
formalin  for  each  1,000  cubic  feet  of  space,  this  apparatus  is  said  to 
yield  a  concentration  of  formaldehyd  of  about  25  grams  to  the  cubic 
meter. 

To  do  away  with  the  use  of  liquid,  a  method  has  been  devised 
which  depends  in  principle  upon  the  breaking  up  by  heat  of  the  solid 
polymer  of  formaldehyd  (tri- 
oxymethylene).  The  apparatus 
(trade  name,  ''Schering's  Para- 
form  Lamp")  as  described  by 
Aronson  ^  consists  of  a  cylindri- 
cal mantle  of  sheet-iron  placed 
upon  a  stand  and  supplied  be- 
low with  an  alcohol  lamp.  Set 
into  the  top  of  the  mantle  is  a 
small  chamber,  into  which  1 
gram  tablets  of  'trioxymethylene 
are  placed.  The  alcohol  lamp, 
so  placed  that  the  wicks  project 

but  slightly — ^to  avoid  overheating — is  lighted,  and  the  formalin  gen- 
erated passes  out  through  slits  in  the  upper  case,  mingling  with  the 
water  vapor  and  other  gases  liberated  by  the  alcohol  flame.  The 
more  modem  devices  have  water-boiler  attachments  to  insure  suffi- 
cient moisture.  Two  tablets  are  sufficient  for  the  fumigation  of  about 
thirty-five  cubic  feet,  and  2  c.c.  of  alcohol  are  filled  into  the  lamp  for 
each  tablet.  One  hundred  to  one  hundred  and  fifty  tablets  are 
usually  enough  for  the  ordinary  room. 

A  simple  method  of  generating  formaldehyd  is  that  which  is  known 
as  the  ''lime  method.'^  In  a  wide  shallow  pan  40  per  cent  formalde- 
hyd solution  (commercial  formaldin)  is  poured  over  quicklime  (CaO). 
The  previous  addition  of  concentrated  sulphuric  acid  to  the  formalin,  in 
proportions  of  one  to  ten,  increases  the  speed  of  formalin  liberation,  and 
aids  in  limiting  poljnnerization.  One  and  one-half  to  two  pounds  (one- 
half  to  one  kilogram)  of  quick-lime  are  used  for  every  500  c.c.  of  the  for- 
malin solution.  The  heat  generated  in  the  slaking  of  the  lime  produces 
volatilization  of  the  formalin. 

A  modification  of  this  method  is  that  of  Schering  ^  in  which  tab- 


1  Aronson,  Zeit.  f .  Hyg.,  xxv,  1897. 

2  Schering,  Hyg.  Rundschau,  1900. 


92  BIOLOGY  AND  TECHNIQUE 

lets  of  paraform  and  unslaked  lime  are  together  laid  into  a  pan  and 
warm  water  is  poured  over  them. 

A  very  simple  method  is  the  potassium  permanganate  method  of 
Evans  and  Russell.^  This  method  depends  upon  the  active  reaction  oc- 
curring when  formalin  and  potassium  permanganate  are  mixed.  Per- 
manganate is  placed  into  a  bucket  and  the  formalin  poured  over  it.  The 
bucket  should  be  large  enough  to  prevent  overflowing  when  the  mixture 
foams.  Special  galvanized  iron  pails  are  made  with  funnel-shaped  tops, 
and  pails  should  be  placed  on  a  piece  of  iron  sheeting  or  bricks  to  prevent 
overheating  of  the  floor;  500  c.c.  of  commercial  formalin  and  250  grams 
of  potassium  manganate  should  be  used  for  every  1,000  cu.  ft. 

The  room  in  which  formaldehyd  has  been  liberated  is  kept  sealed,  in 
the  manner  already  described,  for  at  least  twelve  hours,  after  which  the 
windows  and  doors  are  opened.  The  odor  which  remains  after  formalde- 
hyd fumigation  may  be  removed  by  sprinkling  with  anmionia,  or  by 
the  use  of  some  one  or  another  of  the  various  sorts  of  apparatus  de- 
vised for  the  liberation  of  ammonia. 

For  the  destruction  of  rodents,  hydrocyanic  add  gas  is  used  in  the 
fumigation  of  ships  and  houses.  This  is  of  especial  importance  in  con- 
trolling such  diseases  as  plague.  Recently  Creel,  Faget,  and  Wrightson, 
of  the  United  States  Public  Health  Service,  have  studied  this  method. 
They  found  hydrocyanic  acid  gas  is  more  penetrating,  more  toxic,  and 
more  easily  applied  than  either  sulphur  dioxid  or  caxbon  monoxid.  5  oz. 
of  powdered  potassium  cyanid  per  1,000  cu.  ft.  of  space  was  sufficient  to 
kill  rodents.  The  gas  is  produced  by  dropping  the  potassium  cyanid  into 
sulphuric  acid  of  a  specific  gravity  of  1.84,  or  commercial  grade  66B. 
The  gas  is  as  effective  for  insects  as  it  is  for  rodents.  The  element  of 
danger  to  human  life  must  be  always  considered  in  carrying  out  such 
fumigation,  but  the  writers  referred  to  believe  that  there  is  no  danger 
to  men  entering  a  place  fimiigated  in  this  way  30  minutes  or  longer  after 
the  apertures  have  been  opened.  For  the  fumigation  of  the  hold  of  a 
ship,  Creel,  Faget,  and  Wrightson  ^  use  an  ordinary  wooden  barrel,  into 
the  top  of  which  is  placed  a  large  galvanized  iron  funnel.  The  cyanid,  in 
5-gal.  tins  with  top  removed,  may  be  attached  to  the  funnel  and  dumped 
into  the  sulphuric  acid  by  means  of  a  rope  attached  to  the  tin,  after  the 
barrel  has  been  lowered. 


1  Evans  and  Russell,  Rep.  State  Bd.  Health,  Maine,  1904. 

2  Creel,  Faget,  and  Wrightson,  United  States  Public  Health  Reports,  Vol.  30,  No. 
39,  Dec.  3,  1915. 


CHAPTER  VI 

METHODS  USED  IN  THE  MICROSCOPIC  STUDY  AND  STAINING 

OF  BACTERIA 

MICROSCOPIC   STUDY   OF   BACTERIA 

Bacteria  may  be  studied  microscopically,  in  the  living  and  un- 
stained state,  and,  after  the  application  of  dyes,  in  colored  preparations. 
For  the  manipulation  of  bacteria  for  such  study,  glass  slides  and  cover- 
slips  of  various  design  are  used.  These  must  be  perfectly  clean  if  the 
preparations  are  to  be  of  any  value. ^ 

The  Study  of  Bacteria  in  the  Living  State. — Living  bacteria  may  be 
studied  in  what  is  spoken  of  as  the  "hanging-drop"  preparation. 
For  this  purpose  a  so-called  hollow  slide  is  employed,  in  the  center  of 
which  there  is  a  circular  concavity  about  three-quarters  of  a  centimeter 
to  one  centimeter  in  diameter.  The  preparation  is  manipulated  as 
follows:  If  the  bacteria  are  growing  in  a  fluid  medium  a  drop  of  the 
culture  fluid  is  transferred  to  the  center  of  a  cover-slip.  If  taken  from 
solid  media,  an  emulsion  may  be  made  in  broth  or  in  physiological  salt 
solution,  and  a  drop  of  this  transferred  to  the  cover-slip,  or  the  bac- 
teria may  be  emulsified  in  a  drop  of  salt  solution,  or  broth,'>^irectly  upon 
the  cover-slip.  The  concavity  on  the  slide,  having  first  been  rimmed 
with  vaseline,  by  means  of  a  small  camel's-hair  brush,  the  cover-slip  is 
inverted  over  the  slide  in  such  a  way  that  the  drop  hangs  freely  within 
the  hollow  space.  The  preparation  is  then  ready  for  examination  under 
the  microscope. 

1  Although  the  silicates  of  which  glass  is  composed  are  extremely  stable,  never- 
theless alkaline  silicates  which  are  said  to  separate  out  on  the  surface,  together  with 
grease  and  dirt  left  upon  the  glass  by  handling,  during  blowing  and  cutting,  neces- 
sitate  cleansing  before  use.  This  may  be  accomplished  by  a  variety  of  methods,  A 
simple  one  suitable  for  general  application  is  as  follows:  (1)  The  slides  and  cover- 
slips  are  thrown  singly  into  boihng  water  and  left  there  for  half  an  hour;  (2)  wash 
in  twenty-five  per  cent  sulphuric  acid;  (3)  rinse  in  distilled  water;  (4)  wash  in 
alcohol;  (5)  wipe  with  a  clean  cloth  and  keep  dry  under  a  bell-jar.  Another  method 
convenient  for  routine  use  is  to  immerse,  after  thorough  washing  in  soap-suds 
and  acid^  in  ninety-five  per  cent  alcohol  and  to  leave  in  this  until  the  time  of  use. 

93 


94  BIOI-OGY  AND  TECHNIQUE 

Another  method,  known  as  the  ''  hanging  block  method,"  devised  by 
Hill,^  for  the  study  of  Uving  bacteria  in  soUd  media  is  carried  out  as  fol- 
lows: Nutrient  agar  is  poured  into  a  Petri  dish  and  allowed  to  solidify. 
Out  of  this  layer  a  piece  about  a  quarter  of  an  inch  square  is  cut.  This 
is  placed  on  a  sterile  slide.  The  upper  surface  of  the  agar  block  is  then 
inoculated  with  bacteria  by  surface  smearing,  and  the  preparation 
covered  with  a  sterile  dish  and  allowed  to  dry  for  a  few  minutes  in  the 
incubator.    A  sterile  cover-slip  is  then  dropped  upon  the  surface  of  the 


^ 


Fig.  14. — Hanging  Drop  Preparation. 

block  and  sealed  about  the  edges  with  agar.  Block  and  cover-slip  are 
then  taken  from  the  slide  and  fastened  over  a  moist  chamber  with  paraf- 
fin. The  entire  preparation  can  be  placed  upon  the  stage  of  a  microsocpe. 
This  method  is  especially  designed  for  the  study  of  cell-division. 

Living  bacteria  may  also  be  studied  in  stained  preparations  by  the 
so-called  "  intravital "  method  of  Nakanishi.  Thoroughly  cleaned  slides 
are  covered  with  a  saturated  aqueous  solution  of  methylene-blue.  This 
is  spread  over  the  slide  in  an  even  film  and  allowed  to  dry.  After  drying 
the  slide  should  appear  of  a  trg-nsparent  sky-blue  color.  The  micro- 
organisms which  are  to  be  examined  are  then  emulsified  in  water,  or  are 
taken  from  a  fluid  medium  and  placed  upon  a  cover-slip.  This  is  dropped, 
face  downward,  upon  the  blue  grouni  of  the  slide.  In  this  way  bacteria 
may  be  stained  without  being  subjected  to  the  often  destructive  proc- 
esses of  heat  or  chemical  fixation.  According  to  Nakanishi,  cytoplasm 
is  stained  blue,  while  nuclear  material  assumes  a  reddish  or  purplish 
hue. 

The  Study  of  Bacteria  in  Fixed  Preparations. — Stained  preparations 
of  bacteria  are  best  prepared  upon  cover-slips,  the  process  consisting  of 
the  following  steps :  (1)  Spreading  on  cover-slip;  (2)  drying  in  air;  (3) 
fixing;   (4)  staining;   (5)  washing  in  water;  (6)  blotting;    (7)  mounting. 

(1)  Smearing. — Bacteria  from  a  fluid  medium  are  transferred  in  a 
small  drop  of  the  fluid,  with  a  platinum  loop,  to  a  cover-slip  and  care- 
fully spread  over  the  surface  in  a  thin  film.  If  taken  from  a  solid  medium 
a  small  drop  of  sterile  water  is  first  placed  upon  the  cover-slip  and  the 
bacteria  are  then  in  very  small  quantity  carefully  emulsified  in  this  drop 
with  the  platinum  needle  or  loop  and  spread  in  an  extremely  thin  film. 


HUl,  Jour,  of  Med.  Research,  vii,  1902. 


MICROSCOPIC   STUDY   AND    STAINING  95 

(2)  The  film  is  allowed  to  dry  in  the  air. 

(3)  When  thoroughly  dried,  fixation  is  carried  out  by  passing  the 
preparation,  film  side  up,  three  times  through  a  Bunsen  flame,  at  about 
the  rate  of  a  pendulum  swing.  Fixation  by  heat  in  this  manner  is  most 
convenient  for  routine  work,  but  is  not  the  most  delicate  method,  in- 
asmuch as  the  degree  of,  heat  applied  can  not  be  accurately  controlled. 
The  other  methods  which,  may  be  employed  are  immersion  in  methyl 
alcohol,  formalin,  saturated  aqueous  bichloride  of  mercury,  Zenker^s 
fluid,  or  acetic  acid.  If  chemical  fixatives  are  used,  they  must  be  re- 
moved by  washing  in  water  before  the  stain  is  applied.  If  a  prepara- 
tion is  made  upon  a  slide  instead  of  a  cover-slip,  passage  through  the 
flame  should  be  repeated  eight  or  nine  times. 

(4)  Staining. — The  dyes  used  for  the  staining  of  bacteria  are,  for 
the  greater  part,  basic  anilin  dyes,  such  as  methylene-blue,  gentian- 
violet,  and  fuchsin.  These  may  be  applied  for  simple  staining  in  5 
per  cent  aqueous  solutions  made  up  from  filtered  saturated  alcoholic 
solutions,  or  directly  by  weight.  They  are  conveniently  kept  in  the 
laboratory  as  saturated  alcoholic  solutions.  The  strengths  of  some 
saturated  solutions  are  as  follows: 


Saturated  Solutions  ^  (Stains  Gruebler  or  Merck). 

Fuchsin  (aqueous),  1.5  per  cent. 

Fuchsin  (alcohol  96  per  cent),  3  per  cent. 

Gentian-violet  (aqueous),  1.5  per  cent. 

Gentian-violet  (alcohol  96  per  cent),  4.8  per  cent. 

Methylene-blue  (aqueous),  6.7  per  cent. 

Methylene-blue  (alcohol  96  per  cent),  7  per  cent. 

The  staining  solution,  in  simple  routine  staining,  is  left  upon  the  fixed 
bacterial  film  for  from  one-half  to  one  and  one-half  minutes  according  to 
the  efficiency  of  the  stain  used.  Methylene-blue  is  the  weakest  of  the 
three  stains  mentioned;    gentian-violet  the  strongest. 

(5)  The  excess  stain  is  removed  by  washing  with  water. 

(6)  The  preparation  is  thoroughly  dried  by  a  blotter  or  between 
layers  of  absorbent  paper. 

(7)  A  small  drop  of  Canada  balsam  is  placed  upon  the  film  side  of 
the  dry  cover-slip,  which  is  then  inverted  upon  a  slide.  The  prepara- 
tion is  now  ready  for  microscopical  examination. 

1  After  Wood,  "  Chemical  and  Microscopical  Diagnosis,"  Appendix.    N.  Y.,  1909. 
8 


96  BI0LCX5Y  AND  TECHNIQUE 

The  chemical  principles  which  underlie  the  staining  process  are  still 
more  or  less  in  doubt. ^  Suffice  it  to  say  here  that  most  of  the  dyes  m 
common  use  by  bacteriologists  and  pathologists  are  coal-tar  derivatives 
belonging  to  the  aromatic  series,  all  of  them  containing  at  least  one 
"benzolring"  combined  with  what  Michaelis  terms  a  "chromophore 
group/'  chief  among  which  are  the  nitro-group  (NO2),  the  nitroso-group 
(NO),  and  the  azo-group  (N  =  N) .  Just  what  the  actual  process  of  stain- 
ing consists  in,  is  a  question  about  which  various  opinions  are  held,  some 
believing  that  the  phenomenon  is  purely  chemical,  in  which  a  salt  is 
formed  by  the  combination  of  the  dye  and  the  protoplasm  of  the  cells, 
others  that  there  is  no  such  salt  formation,  and  that  the  process  takes 
place  by  purely  physical  means.  To  support  the  latter  view  it  is  argued 
that  certain  substances  like  cellulose  are  stainable  without  possessing 
the  property  of  salt  formation,  and  that  staining  may  often  be  accom- 
plished without  there  being  a  chemical  disruption  of  the  dye  itself. 
Michaelis  sums  up  his  views  by  stating  that  probably  both  processes 
actually  take  place.  A  dye  stuff,  as  a  whole,  may  enter  into  and  be  de- 
posited upon  a  tissue  or  cell  by  a  process  which  he  speaks  of  as  "  insorp- 
tion."  In  such  a  case  the  coloring  matter  may  be  subsequently  ex- 
tracted by  any  chemically  indifferent  solvent.  On  the  other  hand,  a  dye 
after  being  thus  deposited  upon  or  within  a  cell,  may  become  chemically 
united  to  the  protoplasm  by  the  formation  of  a  salt,  and  in  such  a  case 
the  color  can  be  removed  only  by  agents  which  are  capable  of  decoiv- 
posing  salts,  such  as  free  acids. 

The  staining  power  of  any  solution  may  be  intensified  either  by 
heating  while  staining,  by  prolonging  the  staining  process,  or  by  the 
addition  of  alkalies,  acids,  anilin  oil,  and  other  substances  which  v/ilJ 
be  mentioned  in  the  detailed  descriptions  of  special  staining  methods. 

One  of  the  most  common  examples  of  such  an  intensified  stain 
is  the  so-called  Loeffler's  alkaline  methylene-blue.  This  is  made  up  in 
the  following  way: 

Saturated  alcoholic  solution  of  methylene-blue,    30  c.c. 
1  :  10,000  solution  potassium  hydrate  in  water,  100  c.c. 

Another  solution  designed  with  a  similar  purpose  is  the  Koch-Ehrlich 
anilin-water  solution.  Anilin  oil,  one  part,  is  shaken  up  with  dis- 
tilled water,  nine  parts;  after  thorough  shaking,  the  mixture  is  filtered 

1  For  comprehensive  reviews  of  the  subject,  the  reader  is  referred  to  dissertations 
such  as  those  of  Mann  ("  Physiol.  Hist.  Methods  and  Theory,"  Oxford,  1902)  and 
of  Michaelis  ("  Einfiihrung  in  die  Farbstoffchemie,"  etc.,  Berlin^  1902). 


MICROSCOPIC   STUDY   AND    STAINING  ^7 

through  a  moist  filter  paper  until  perfectly  clear.  A  saturated  alco- 
holic solution  of  either  fuchsin  or  gentian-violet  is  added  to  this  anilin 
water  in  proportions  of  about  one  to  ten  or  until  a  slightly  iridescent 
pellicle  appears  upon  the  surface  of  the  solution. 

An  extremely  useful  and  very  strong  staining  solution  is  the  Ziehl 
carhol-fuchsin  solution,  rnade  up  as  follows :  ^ 

Fuchsin  (basic) ^rVxV.^-r*^.. .  .yv>  .>.yV^rV??^A< 1  gm. 

1.  Alcohol  (absolute) 10  c.c. 

Five  per  cent  carbolic  acid     100  c.c.   y**^  ^1H 

To  make  up  this  staining  solution,  mix  90  c.c.  of  a  five  per  cent  aque- 
ous solution  of  carbolic  acid  with  10  c.c.  of  saturated  alcoholic  basic 
fuchsin. 

It  may  also  be  made  up  as  follows: 

Weigh  out 

Basic  fuchsin 1  gram 

Carbolic  acid 5  grams 

Dissolve  in 

Distilled  water 100  c.c. 

Filter  and  add 

Absolute  alcohol 10  c.c. 

SPECIAL   STAINING    METHODS 

Spore  Stains. — Abbott's  Method.^ — Cover-slips  are  smeared  and 
fixed  by  heat  in  the  usual  manner. 

Cover  with  Loeffler's  alkaline  methylene-blue  and  heat  the  stain 
until  it  boils,  repeat  the  heating  at  intervals  but  do  not  boil  continuously. 
Keep  this  up  for  one  minute. 

Rinse  in  water. 

Decolorize  with  a  mixture  of  alcohol  eighty  per  cent  98  c.c.  and  nitric 
acid  2  c.c,  until  all  blue  has  disappeared. 

Rinse  in  water. 

Dip  from  three  to  five  seconds  in  saturated  alcoholic  solution  of 
eosin  10  c.c,  and  water  90  c.c 

Rinse  in  water,  blot,  and  mount  in  balsam. 

By  this  method  the  spores  are  stained  blue,  the  bodies  of  the  bacilli 
are  stained  pink. 

1  Ziehl,  Deut.  med.  Woch.,  1882.  ^Abbott,  "Prin.  of  Bact.,"  Phila.,  1905- 


98  BIOLOGY    AND   TECHNIQUE 

Moeller's  Method.^ — Cover-slips  are  prepared  as  usual  and  fixed 
in  the  flame. 

Wash  in  chloroform  for  two  minutes. 

Wash  in  water. 

Cover  with  five  per  cent  chromic  acid  one-half  to  two  minutes. 

Wash  in  water.  Invert  and  float  cover-slip  on  carbol-fuchsin  solu- 
tion in  a  small  porcelain  dish  and  heat  gently  with  a  flame  until  it  steams; 
continue  this  for  three  to  five  minutes.  (This  step  can  also  be  done  by 
covering  the  coyer-glass  with  carbol-fuchsin  and  holding  over  flame.) 

Decolorize  with  five  per  cent  sulphuric  acid  five  to  ten  seconds. 

Wash  in  water. 

Stain  with  aqueous  methylene-blue  one-half  to  one  minute.  By 
this  method  spores  will  be  stained  red,  the  body  blue. 

Capsule  Stains. — Welch's  Method.^ — Cover-slips  are  prepared  as 

usual  but  dried  without  heat. 

Cover  with  glacial  acetic  acid  for  a  few  seconds.  Pour  off  acetic  acid 
and  cover  with  anilin  water  gentian-violet,  renewing  stain  repeatedly 
until  all  acid  is  removed.  This  is  done  by  pouring  the  stain  on  and  off 
three  or  four  times  and  then  finally  leaving  it  on  for  about  three  minutes. 

Wash  in  two  per  cent  salt  solution  and  examine  in  this  solution. 

Hiss'  Methods.^ — (1)  Copper  Sulphate  Method. — Cover-slip  prepara- 
tions are  made  by  smearing  the  organisms  in  a  drop  of  animal  serum, 
preferably  beef-blood  serum. 

Dry  in  air  and  fix  by  heat. 

Stain  for  a  few  seconds  with — 

Saturated  alcoholic  solution  of  fuchsin  or  gentian-violet  5  c.c,  in 
distilled  water  95  c.c. 

The  cover-slip  is  flooded  with  the  dye  and  the  preparati-jn  held  for  a 
second  over  a  free  flame  until  it  steams. 

Wash  off  dye  with  twenty  per  cent  aqueous  copper  sulphate  solution. 

Blot  (do  not  wash). 

Dry  and  mount. 

By  this  method  permanent  preparations  are  obtained,  the  capsule 
appearing  as  a  faint  blue  halo  around  a  dark  purple  cell  body. 

Huntoon's  Capsule  Stain  (applicable  only  to  cultures,  not  to 
animal  exudates). — This  depends  upon  the  precipitating  action  of 
lactic  acid  on  nutrose.     Requires  two  solutions. 

» Moeller,  Cent.  f.  Bakt.,  I,  x,  1891. 

2  Welch,  Johns  Hopkins  Hosp.  Bull.,  1892,  iii,  81. 

'  Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exper.  Med.,  vi,  1905. 


MICROSCOPIC  STUDY  AND  STAINING  99 

1.  Diluent. — 3  per  cent  solution  of  nutrose  in  distilled  water;  place  in  Arnold  one 
hour,  add  a  small  amount  of  carbolic  as  preservative,  and  allow  to  settle. 

2.  Stain  and  fixative. — 2  percent  carbolic,  100  c.c;  concentrated  lactic  acid,  0.5 
c.c;  1  per  cent  acetic  acid,  1  c.c;  saturated  alcoholic  solution  basic  fuchsin,  1  c.c; 
carbol  fuchsin,  1  c.c 

As  to  the  dye  employed,  most  anything  but  methylene  blue  or  Bismarck  brown 
may  be  used.  Methyl  violet  gives  the  most  beautiful  results  but  is  not  permanent 
and  will  not  photograph.  I  have  found  the  above  mixture  the  best  for  classroom 
work. 

Make  a  thin  film,  employing  solution  1  as  diluent.  Dry  in  air.  Do 
not  fix.  Cover  with  stain  3p  seconds.  Wash  in  water,  dry,  and 
examine. 

Buerger's  Method.* — Cover-slip  preparations  are  made  by  smear- 
ing in  serum  as  in  Hiss'  method. 

As  the  edges  of  the  smear  begin  to  dry,  pour  over  it  Zenker's  fluid 
(without  acetic  acid)  and  warm  in  flame  for  three  seconds. 

(Zenker's  fluid  is  composed  of  potassium  bichromate  2.5  gm., 
sodium  sulphate  1  gm.,  water  100  c.c,  saturated  with  bichloride  of 
mercury.) 

Wash  in  water. 

Flush  with  ninety-five  per  cent  alcohol. 

Cover  with  tincture  of  iodin,  U.  S.  P.,  one  to  three  minutes. 

Wash  with  ninety-five  per  cent  alcohol. 

Dr}^  in  the  air. 

Stain  v/ith  anilin  water  gentian-violet  two  to  five  seconds. 

Wash  with  two  per  cent  salt  solution. 

Mount  and  examine  in  salt  solution. 

Wads  worth's  Method.^ — Wadsworth  has  devised  a  method  of 
staining  capsules  which  depends  upon  the  fixation  of  smears  with  forma- 
lin. After  such  fixation  capsules  may  be  demonstrated  both  with  simple 
stains  and  by  Gram's  method.    The  technique  is  as  follows : 

Smear  preparations,  made  as  usual,  are  treated  as  follows: 

1.  Formalin,  40  per  cent,  two  to  five  minutes. 

2.  Wash  in  water,  five  seconds. 

Simple  Stain.  Differkntial  Stain  (Gram's  Method). 

3.  Ten  per  cent  aqueous  gentian-violet.  3.  Anilin  gentian-violet,  two  minutes. 

4.  Wash  water,  five  seconds.  4.  Iodin  solution,  two  minutes. 

5.  Dry,  moimt  in  balsam.  5.  Alcohol,  95  per  cent,  decolorize. 

6.  Fuchsin,  dilute  aqueous  solution. 

7.  Wash  water,  two  seconds. 

8.  Dry,  mount  in  balsam. 

1  Buerger,  Med.  News,  Dec,  1904  «  Wadsworth,  Jour.  Inf.  Dis.,  1906. 


100  BIOLOGY    AND    TECHNIQUE 

It  is  important  that  the  formalin  be  fresh  and  the  exposure  to 
water  momentary.  When  decolorizing  in  the  Gram  method,  strong 
alcohol  only  should  be  used.  Wadsworth  also  found  that  encapsulated 
pneumococci  could  be  demonstrated  in  celloidin  sections  of  pneumonic 
lesions  hardened  in  strong  formalin.  The  lungs  should  be  distended  with 
the  formalin  or  the  lesions  cut  in  very  thin  bits,  hardened,  dehydrated, 
embedded,  and  cut  in  the  usual  way.  The  celloidin  sections  may  be  fixed 
on  the  slides  by  partially  dissolving  the  celloidin  in  alcohol  and  ether 
and  setting  the  celloidin  quickly  in  water  before  staining.  Failure  to 
obtain  pneumococci  encapsulated  in  such  sections  is  usually  due  to 
improper  or  inadequate  fixation  in  the  formalin. 

The  differential  method  employed  by  Wadsworth  for  tissue  staining 
is  as  follows: 

1.  Fix  in  formalin  forty  per  cent,  two  to  five  minutes. 

2.  Wash  in  water. 

3.  AnUin  gentian-violet,  two  minutes. 

4.  lodin  solution,  two  minutes. 

5.  Alcohol,  ninety-five  per  cent,  decolorize. 

6.  Eosin  alcohol,  counterstain. 

7.  Clear  in  oil  of  origanum. 

8.  Mount  in  balsam. 

Flagella  Stains. — All  flagella  stains,  in  order  to  be  successful,  neces- 
sitate particularly  clean  cover-slip  preparations,  best  made  from  young 
agar  cultures  emulsified  in  sterile  salt  solution.  Scrupulous  care  should 
be  exercised  in  cleaning  the  glassware  used. 

Loeffler's  Method.^ — The  preparation  is  dried  in  the  air  and  fixed 
by  heat.    It  is  then  treated  with  the  following  mordant  solution: 

Twenty  per  cent  aqueous  tannic  acid 10  parts. 

Ferrous  sulphate  aq.  sol.  saturated  at  room  temperature .  5  parts. 
Saturated  alcoholic  f uchsin  solution 1  part. 

This  solution,  which  should  be  freshly  filtered  before  using,  is 
poured  over  the  cover-glass  and  allowed  to  remain  there  for  on«^-half 
to  one  minute,  during  which  time  it  should  be  gently  heated,  but  not 
allowed  to  boil. 

Wash  thoroughly  in  water. 

Stain  with  five  per  cent  anilin  water  fuchsin  or  anilin  water  gen- 


» Loeffler,  Cent.  f.  Bakt.,  I,  vi,  1889. 


MICROSCOPIC    STUDY   AND    STAINING  101 

tian-violet  made  slightly  alkaline  by  the  addition  of  one-tenth  per  cent 
sodium  hydrate. 

The  stain  should  be  filtered  directly  upon  the  cover-slip.  Warm 
gently  and  leave  on  for  one  to  two  minutes.  Wash  in  water.  Mount  in 
balsam. 

Van  Ermengem's  Method.^ — This  method  requires  the  preparation 
of  three  solutions. 

(1)  Twenty  per  cent  tannic  acid  solution 60  c.c. 

Two  per  cent  osmic  acid  solution 30  c.c. 

Glacial  acetic  acid 4-5  drops. 

The  cover-slip  carrying  the  fixed  preparation  is  placed  in  this  solu- 
tion for  one  hour  at  room  temperature,  or  for  five  minutes  at  100°  C. 
(boiling) . 

Wash  in  water. 

Wash  in  absolute  alcohol. 

Immerse  the  cover-slip  for  one  to  three  seconds  in 

(2)  Silver  nitrate,  0.25-0.5  per  cent  solution. 
Without  washing,  transfer  to 

(3)  Gallic  acid 5  gm. 

Tannic  acid 3    " 

Fused  potassium  acetate 10    " 

Distilled  water 350  c.c. 

Immerse  in  this  for  a  few  minutes,  moving  the  cover-slip  about. 

Return  to  the  silver  nitrate  solution  until  the  preparation  turns 
black. 

Wash  thoroughly  in  water. 

Blot  and  mount. 

Smith's  Modification  of  Pitfield's  Method.^ — A  saturated  solu- 
tion of  bichloride  of  mercury  is  boiled  and  is  poured  while  still  hot  into  a 
bottle  in  which  crystals  of  ammonia  alum  have  been  placed  in  quantity 
more  than  sufficient  to  saturate  the  fluid.  The  bottle  is  then  shaken  and 
allowed  to  cool.  Ten  c.c.  of  this  solution  are  added  to  10  c.c.  of  freshly 
prepared  ten  per  cent  tannic  acid  solution.  To  this  add  5  c.c.  carbol- 
fuchsin  sohition.     Mix  and  filter. 

To  stain,  filter  the  above  mordant  directly  upon  the  fixed  cover-slip 

1  Van  Ermengem,  Cent.  f.  Bakt.,  I,  xv,  1894. 

2  .SmtV/i,  Brit.  Med.  Jour.,  I,  1901,  p.  205. 


102  BIOLOGY  AND  TECHNIQUE 

preparation.  Heat  gently  for  three  minutes,  but  do  not  allow  to  boil. 
Wash  in  water  and  stain  with  the  following  solution: 

Saturated  alcoholic  solution  gentian-violet 1  c.c. 

Saturated  solution  ammonia  alum 10  c.c. 

Filter  the  stain  directly  upon  the  preparation  and  heat  for  three  or  four 
minutes.    Wash  in  water,  dry,  and  mount  in  balsam. 

Differential  Stains. — Gram's  Method.^ — By  this  method  of  staining, 
which  is  extremely  important  in  bacterial  differentiation,  bacteria  are 
divided  into  those  which  retain  the  initial  stain  and  those  which  are 
subsequently  decolorized  and  take  the  counterstain.  The  former  are 
often  spoken  of  as  the  Gram-positive,  the  latter  as  Gram-negative 
bacteria. 

Preparations  are  made  on  cover-slips  or  slides  in  the  usual  way. 

The  preparation  is  then  covered  with  an  anilin  gentian-violet  solu- 
tion which  is  best  made  up  freshly  before  use. 

The  staining  fluid  is  made  up,  according  to  Gram's  original  direc- 
tions,^ as  follows: 

Five  c.c.  of  anilin  oil  are  shaken  up  thoroughly  with  125  c.c.  of  dis- 
tilled  water.    This  solution  is  then  filtered  through  a  moist  filter  paper. 

To  108  c.c.  of  this  anilin  water,  add  12  c.c.  of  a  saturated  alcoholic 
solution  of  gentian- violet.  The  stain  acts  best  when  twelve  to  twenty- 
four  hours  old,  but  may  be  used  at  once.  It  lasts,  if  well  stoppered,  for 
three  to  five  days.  A  more  convenient  and  simple  method  of  making  up 
the  stain  is  as  follows: 

To  10  c.c.  of  distilled  water  in  a  test  tube  add  anilin  oil  until  on 
shaking  the  emulsion  is  opaque;  roughly,  one  to  ten.  Filter  this  through 
a  wet  paper  until  the  filtrate  is  clear.  To  this  add  saturated  alcoholic 
solution  of  gentian- violet  until  the  mixture  is  no  longer  transparent, 
and  a  metallic  film  on  the  surface  indicates  saturation.  One  part  of 
alcohoHc  saturated  gentian-violet  to  nine  parts  of  the  anilin  water 
will  give  this  result.  This  mixture  may  be  used  immediately  and  lasts 
two  to  five  days  if  kept  in  a  stoppered  bottle. 

Cover  the  preparation  with  this;  leave  on  for  5  minutes.  Pour  off 
excess  stain  and  cover  with  Gram's  iodin  solution  for  2  to  3  minutes. 

lodin. 1  gm. 

Potassium  iodid _  .     2  gm. 

Distilled  water 300  c.c. 


1  Gram,  Fortschr.  d.  Med.,  ii,  1884.  2  Oraniy  loc.  cit- 


MICROSCOPIC  STUDY  AND  STAINING  103 

Decolorize  with  ninety-seven  per  cent  alcohol  until  no  further  traces 
of  the  stain  can  be  washed  out  of  the  preparation.  This  takes  usually 
thirty  seconds  to  two  minutes,  according  to  thinness  of  preparation. 

Wash  in  water. 

Counterstain  with  an  aqueous  contrast  stain,  preferably  Bismarck 
brown.i 

Paltauf's  Modification  of  Gram's  Stain.^ — The  staining  fluid  as 
prepared  by  this  modification  possesses  the  advantage  of  retaining  its 
staining  power  for  a  longer  period  than  does  the  anilin-water  gentian- 
violet  described  in  the  original  method. 

The  staining  fluid  is  prepared  as  follows: 

3-5  c.c.  anilin  oil  are  added  to 
90  c.c.  distilled  water  and 
7  c.c.  absolute  alcohol. 

This  mixture  is  thoroughly  shaken  and  filtered  through  a  moist 
filter  paper  imtil  clear.     Then  add: 

Gruebler's  gentian-violet  2  gm. 

The  fluid  should  stand  twenty-four  hours,  during  which  a  precipi- 
tate forms.     This  is  filtered  before  use. 

This  gentian-violet  solution  retains  its  staining  power  for  from  4  to  6 
weeks.    It  is  good  only  when  a  metallic  luster  develops  on  the  surface. 

It  is  used  in  the  following  way:  Spreads  on  cover-slips  or  slides  are 
dried  and  fixed  as  usual. 

Then  apply: — 

Anilin-water  gentian-violet  (as  above),  three  minutes. 

Gram's  iodin  solution,  two  minutes. 

Absolute  alcohol  (with  stirring),  thirty  seconds 

Counterstain,  without  washing  in  water,  in  aqueous  fuchsin  or  in 
weak  carbol-fuchsin. 

Sterling's  Modification  of  Gram's  Method. — 2  c.c.  anilin  oil  + 
10  c.c.  95%  alcohol.  Shake  and  add  88  c.c.  distilled  water.  5  grams 
gentian  violet  are  ground  in  a  mortar  and  the  anilin  solution  added 
slowly  while  grinding.  Filter.  This  solution  keeps,  and  stains  in  one- 
half  to  one  minute. 


1  To  make  up  Bismarck  brown  solution,  prepare  a  saturated  aqueous  solution  of 
the  powdered  dye  by  heating.    Cool  and  filter.    Dilute  1  to  10  with  distilled  water. 

2  iSharnosky,  Proc.  N.  Y.  Pathol.  Soc,  Oct.,  1909,  n.  s.,  ix,  5. 


104 


BIOLOGY  AND  TECHNIQUE 


Classification    of   the    Most    Important    Pathogenic    Bacteria 
According  to  Gram's  Stain. 


Gram-positive. 

(Retain  the  Gentian-violet.) 

Micrococcus  pyogenes  aureus 

Micrococcus  pyogenes  albus 

Streptococcus  pyogenes 

Micrococcus  tetragenus 

Pneumococcus 

Bacillus  subtilis 

Bacillus  anthracis 

Bacillus  diphtherise 

Bacillus  tetanus 

Bacillus  tuberculosis  and  other 

acid-fast  bacilli 
Bacillus  aerogenes  capsulatus 
Bacillus  botulinus 


Gram-negative. 

{Take  Counter  stain.) 

Meningococcus 
Gonococcus 

Micrococcus  catarrhalis 
Bacillus  coli 
Bacillus  dysenterisB 
Bacillus  typhosus 
Bacillus  paratyphosus 
Bacillus  fecalis  alkaligenes 
Bacillus  enteritidis 
Bacillus  proteus  (proteus) 
Bacillus  mallei 
Bacillus  pyocyaneus 
Bacillus  influenzae 
Bacillus  mucosus  capsulatus 
Bacillus  pestis 
Bacillus  maligni  oedematis 
Spirillum  cholersB 
Bacillus  Koch- Weeks 
Bacillus  Morax-Axenfeld 


Stains  for  Acid-Fast  Bacteria. — These  methods  of  staining  are  chiefly 
useful  in  the  demonstration  of  tubercle  bacilli.  These  bacteria  because 
of  their  waxy  cell  membranes  are  not  easily  stained  by  any  but  the  most 
intensified  dyes,  but  when  once  stained,  retain  the  color  in  spite  of  ener- 
getic decolorization  with  acid.  For  this  reason  they  are  known  as  acid- 
fast  bacilli.  The  first  method  devised  for  the  staining  of  tubercle 
and  allied  bacilli  was  that  of  Ehrlich. 

Ehrlich  Method.* — ^This  method  is  now  rarely  used.  Cover-slip 
preparations  are  prepared  as  usual  and  fixed  by  heat. 

Stain  with  anilin  water  gentian-violet,  hot,  three  to  five  minutes, 
or  twenty-four  hours  at  room  temperature. 


^Ehrlich,  Deut.  med,  Woch,.  1882. 


MICROSCOPIC   STUDY   AND    STAINING  105 

Decolorize  with  thirty-three  per  cent  nitric  acid  one-half  to  one 
minute. 

Treat  with  sixty  per  cent  alcohol,  until  no  color  can  be  seen  to  come 
off. 

Counterstain  with  aqueous  methylene-blue. 

Rinse  in  water,  dry,  and  mount. 

Ziehl-Neelson  Method.  1 — Thin  smears  are  made  upon  cover- 
slips  or  slides. 

Fix  by  heat. 

Stain  in  carbol-fuchsin  solution  as  given  on  page  97.  The  slide 
or  cover-slip  may  be  flooded  with  the  stain,  and  this  gently  heated  with 
the  flame  until  it  steams,  or  else  the  cover-slip  may  be  inverted  upon 
the  surface  of  the  staining  fluid,  in  a  porcelain  dish  or  watch-glass,  and 
this  heated  until  it  steams.  This  is  continued  for  three  to  five  min- 
utes. Decolorize  with  either  five  per  cent  nitric  acid,  five  per  cent 
sulphuric  acid,  or  one  per  cent  hydrochloric  acid  for  three  to  five 
seconds.  The  treatment  with  the  acid  is  continued  until  subsequent 
washing  with  water  will  give  only  a  faint  pink  color  to  the  preparation. 

Wash  with  ninety  per  cent  alcohol  until  no  further  color  can  be  re- 
moved. If,  after  prolonged  washing  with  alcohol,  a  red  color  still  re- 
mains in  very  thick  places  upon  the  smear,  while  the  thin  areas  appear 
entirely  decolorized,  this  may  be  disregarded. 

Wash  in  water  and  counterstain  in  aqueous  methylene-blue  for 
one-half  to  one  minute. 

Rinse  *n  water,  dry,  and  mount. 

By  this  method  the  tubercle  bacilli  are  colored  red,  other  bacteria 
and  cellular  elements  which  may  be  present  are  stained  blue. 

Gabbet's  Method.^ — Gabbet  has  devised  a  rapid  method  in  which 
the  decolorization  and  counterstaining  are  accomplished  by  one  solu- 
tion. The  specimen  is  prepared  and  stained  with  carbol-fuchsin  as  in 
the  preceding  method.  It  is  then  immersed  for  one  minute  directly  in 
the  following  solution: 

Methylene-blue 2  gms. 

Sulphuric  acid  25  per  cent  (sp.  gr.  1018) 100  c.c. 

Then  rinse  in  water,  dry,  and  mount. 

This  method,  while  rapid  and  very  convenient,  is  not  so  reliable  as 
the  Ziehl-Neelson  method. 

»  Ziehl,  Deut.  med.  Woch.,  1882;  Keelson,  Deut.  med.  Woch.,  1883. 
« Gahhet,  Lancet,  1887. 


106  BIOLOGY  AND  TECHNIQUE 

Pappenheim^s  Method/ — ^The  method  of  Pappenheim  is  devised 
for  the  purpose  of  differentiating  between  the  tubercle  bacillus  and  the 
smegma  bacillus.  Confusion  may  occasionally  arise  between  these  two 
microorganisms,  especially  in  the  examination  of  urine  where  smegma 
bacilli  are  derived  from  the  genitals,  and  less  frequently  in  the  examina- 
tion of  sputum  where  smegma  bacilli  may  occasionally  be  mixed  with 
the  secretions  of  the  pharynx  and  throat. 

Preparations  are  smeared  and  fixed  by  heat  in  the  usual  way. 

Stain  with  hot  carbol-fuchsin  solution  for  two  minutes. 

Pour  off  dye  without  washing  and  cover  with  the  following  mixture: 

Corallin  (rosolic  acid)  1  gm. 

Absolute  alcohol 100  c.c. 

Methylene-blue  added  to  saturation 

Add  glycerin  2 20  c.c. 

This  mixture  is  poured  on  and  drained  off  slowly,  the  procedure  being 
repeated  four  or  five  times,  and  finally  the  preparation  is  washed  in 
water.  The  combination  of  alcohol  and  rosolic  acid  decolorizes  the 
smegma  bacilli,  but  leaves  the  tubercle  bacilli  stained  bright  red. 

BuNGE  AND  Trautenroth  Method.^ — This  method  is  designed  to 
differentiate  between  the  tubercle  and  smegma  bacilli. 

Smear  and  fix  by  heat  in  the  usual  way. 

Wash  with  absolute  alcohol  to  remove  fat. 

Treat  with  five  per  cent  chromic  acid  for  fifteen  minutes. 

Wash  in  several  changes  of  water. 

Stain  with  hot  carbol-fuchsin  for  five  minutes. 

Decolorize  with  sixteen  per  cent  sulphuric  acid  for  three  minutes. 

Counterstain  with  alcoholic  methylene-blue  for  five  minutes. 

Wash  in  water,  dry,  and  mount. 

By  this  method  the  tubercle  bacillus  remains  red,  the  smegma  bacil- 
lus is  decolorized. 

Baumgarten's  Method.'* — This  method  is  recommended  by  the 
author  for  differentiation  between  the  bacillus  of  tuberculosis  and  the 
bacillus  of  leprosy  and  depends  upon  the  fact  that  the  tubercle  bacillus 
is  less  easily  stained  than  Bacillus  leprae. 

Smears  are  prepared  and  fixed  by  heat  in  the  usual  way. 


^  Pappenheim,  Berl.  klin.  Woch.,  1898. 

2  The  glycerin  is  added  after  the  other  constituents  have  been  mixed. 

3  Bunge  und  Trautenroth,  Fortschr.  d.  Med.,  xiv,  1896. 
5  Baumgarten,  Zeit.  f.  wissensch.  Mikrosk.,  1,  1884. 


MICROSCOPIC    STUDY    AND    STAINING  107 

Stain  in  dilute  alcoholic  fuchsin  for  five  minutes. 

Decolorize  for  twenty  seconds  in  alcohol,  ninety-five  per  cent,  ten 
parts,  nitric  acid  one  part. 

Wash  in  water. 

Counterstain  in  methylene-blue. 

Wash  in  water,  dry,  and  mount. 

The  tubercle  bacillus  should  be  blue  and  the  bacillus  of  leprosy  red. 

Special  Stains  for  Polar  Bodies.— These  staining  methods  are  designed 
to  bring  into  view  polar  bodies  as  found,  for  instance,  in  the  bacilli  of 
diphtheria  and  plague. 

Neisser's  Method.^ — Smear  and  fix  in  the  usual  manner. 

Stain  for  two  to  five  seconds  in  the  following  solution : 

Methylene-blue 1  gm. 

Absolute  alcohol 20  c.c. 

Glacial  acetic  acid  50  c.c. 

Distilled  water 1,000  c.c. 

Wash  in  water. 

Counterstain  in  two  per  cent  aqueous  Bismarck  brown  solution  for 
five  seconds. 

B}'  this  method  polar  bodies  are  stained  blue,  while  the  bacillary 
bodies  are  stained  brown. 

Roux's  Method.^ — Two  solutions  are  necessary. 

(1)  Dahlia  violet 1  gm.  . 

Alcohol  90  per  cent 10  c.c. 

Aqua  destillata  ad 100  c.c. 

(2)  Methyl-green 1  gm. 

Alcohol  90  per  cent 10  c.c. 

Aqua  destillata  ad *. 100  c.c. 

Before  use,  one  part  of  solution  No.  1  is  mixed  with  three  parts  of 
solution  No.  2.  The  preparation  is  stained  with  the  mixture  for  two 
minutes  in  the  cold. 

Polychrome  Stains. — The  various  polychrome  stains  are  of  value  to 
the  bacteriologist  chiefly  for  the  staining  of  pus  and  exudates  where  the 
relation  of  bacteria  to  cellular  elements  is  to  be  demonstrated.  They 
are  also  extremely  useful  in  the  study  of  fixed  specimens  of  protozoan 
parasites.    Th9r3  is  a  large  number  of  these  stains  in  use;   a  few  only, 

1  Neisser,  Zeit.  f.  Hyg.,  xxiv,  1897. 

'  Roux  and  Yersin,  Annal.  de  I'inst.  Past.,  1890. 


108  BIOLOGY  AND  TECHNIQUE 

however,  can  be  given  here.  In  principle,  all  these  stains  depend  upon  a 
combination  of  eosin  and  methylene-blue,  these  elements  staining  not 
only  as  units,  but  acting  together  in  combination.  One  and  the  same 
solution,  therefore,  contains  at  least  three  elements  which  color  the 
various  structures  of  the  preparation  selectively. 

Jenner's  Method.^ — This  stain,  because  of  its  simplicity,  is  useful 
for  routine  use.  It  is  made  up  as  follows:  Equal  parts  of  eosin  (Gruebler, 
"  W.  G.")  one  and  two-tenths  per  cent  aqueous  solution,  and  methylene- 
blue  (medicinal,  Gruebler)  one  per  cent  aqueous  solution,  are  mixed  and 
allowed  to  stand  for  twenty-four  hours.  A  coarse  granular  precipitate 
is  formed  which  appears  dark,  with  a  metallic  luster  on  its  surface.  This 
is  separated  by  filtration  and  washed  with  distilled  water  until  the  fil- 
trate appears  almost  clear. 

To  make  up  the  stain  0.5  gram  of  the  dry  precipitate  is  dissolved 
in  100  c.c.  of  methyl  alcohol. 

In  using  the  stain,  preparations  are  not  fixed,  but  simply  dried  in 
the  air  and  immersed  in  the  stain  for  one  to  two  minutes.  After  this, 
wash  in  distilled  water  and  examine. 

Wright's  Modification  of  Leishman's  Method.^ — A  one  per  cent 
solution  of  methylene-blue  (Gruebler)  in  five-tenths  per  cent  solution  of 
sodium  bicarbonate  in  distilled  water  is  steamed  in  a  sterilizer  at  100° 
C.  for  one  hour.  After  this  has  cooled,  a  one-tenth  per  cent  aqueous 
solution  of  eosin  (Gruebler,  W.  G.)  is  added  until  a  metallic  scum  ap- 
pears on  the  surface  of  the  mixture.  (About  five  parts  of  eosin  solution 
to  one  of  methylene-blue  is  necessary.)  The  precipitate  which  forms  is 
collected  by  filtration,  dried,  and  a  saturated  solution  then  made  in 
methyl  alcohol.  This  is  filtered  and  diluted  with  one-quarter  its  bulk 
of  methyl  alcohol. 

To  stain,  cover  the  dried  preparation  with  the  stain  for  one  to 
one  and  one-half  minutes.  Dilute  by  dropping  upon  the  stain  distilled 
water  from  a  pipette  until  a  metallic  film  appears  upon  the  top.  Leave 
this  on  for  three  to  fifteen  minutes.    Wash  in  distilled  water. 

Giemsa's  Method.^ — The  method  of  Giemsa  is  really  a  modification 
of  the  Romano wsky  method.  It  is  widely  applicable,  being  of  great 
value  in  the  staining  of  the  Spirochsete  pallida,  Vincent's  spirilla,  pro- 
tozoa, and  Negri  bodies.    The  stain  has  been  modified  several  times  by 


1  Jenner,  Lancet,  i,  1889. 

2  Wright,  Jour.  Med.  Research,  ii,  1902. 
«  Oiemsa,  Cent.  f.  Bakt.,  I,  xxxvii,  1904. 


MlCHOSCOPlC    STUDY   AND    STAINING  109 

its  originator,  the  following  being  the  formula  given  by  him  in  1904: 
The  substance  referred  to  as  azur  II  and  purchasable  under  that  name, 
consists  of  pure  methylenazur  chloralhydrate  combined  with  an  equal 
quantity  of  methylene-blue  chloralhydrate.  The  substance  referred  to 
as  azur  Il-eosin  is  a  combination  of  this  substance  with  eosin. 
The  staining  fluid  is  made  up  as  follows:^ 

Azur  Il-eosin 3  gms. 

Azur  II 8  gms. 

This  mixture  is  thoroughly  dried  over  sulphuric  acid  in  a  desiccator^ 
finely  powdered,  and  rubbed  through  a  fine  sieve.  It  is  then  dissolved  in 
250  gms.  of  C.  P.  glycerin  (Merck),  at  60°  C.  To  this  ic  added  methyl 
alcohol  (Kahlbaum)  250  c.c,  previously  warmed  to  60°  C.  This  mix- 
ture is  well  shaken  and  allowed  to  stand  at  room  temperature  for 
twenty-four  hours.    The  mixture  is  now  ready  for  use. 

For  use  10  c.c.  of  distilled  water  are  poured  into  a  test  tube  and 
one  to  two  drops  of  a  one  per  cent  potassium  carbonate  solution  are 
added.  Ten  drops  of  the  staining  solution  described  above  (one  drop  to 
the  c.c.)  are  mixed  with  this  slightly  alkaline  water.  The  preparation 
which  is  to  be  stained  is  fixed  in  methyl  alcohol,  dried,  and  covered  with 
the  diluted  staining  solution.  For  the  staining  of  protozoa  and  ex- 
udates containing  bacteria,  ten  to  fifteen  minutes  are  sufficient.  For 
the  staining  of  Negri  bodies  or  Spirochete  pallida,  one  or  more  hours 
of  staining  should  be  employed.  After  staining,  wash  in  running  tap 
water  and  blot. 

Wood's  Method.^ — Wood  has  devised  a  simple  staining  method 
based  on  the  principles  of  the  Giemsa  stain,  in  which  azur  II  and  eosin 
may  be  used  in  separate  solutions.  Preparations  are  fixed  in  strong 
methyl  alcohol  for  five  minutes  and  are  then  stained  in  a  0.1  per  cent 
aqueous  solution  of  eosin  until  the  preparation  is  pink.  The  eosin  is 
then  poured  off  and  the  preparation  is  covered  with  a  0.25  per  cent 
aqueous  solution  of  azur  II  for  one-half  to  two  minutes.  Following  this, 
it  is  washed  in  tap  water  and  dried  by  blotting. 

When  an  intense  stain  is  desired,  the  solution  of  eosin  and  azur  II 
may  be  flooded  over  the  preparation  together,  using  an  excess  of  azur 
II.  They  are  then  left  on  from  five  to  ten  minutes.  At  the  end  of  this 
time  washing  and  drying  as  before  completes  the  process. 

» It  is  best  not  to  attempt  to  make  up  the  undiluted  staining  fluid,  since  this  is 
purchasable  under  the  name  of  "  Giemsa  Losung  f  iir  Romanowsky  Farbung." 
2  Wood,  Med.  News,  83,  1903. 


110  BIOLOGY  AND  TECHNIQUE 

The  Staining  of  Bacteria  in  Tissues. -^The  preparation  of  tissue  for 
bacterial  staining  is,  in  general,  the  same  as  that  employed  for  purposes 
of  cellular  studies,  in  histology.  For  bacteriological  studies  the  most 
useful  fixative  is  alcohol;  other  fixations,  such  as  that  by  formalin, 
Zenker's  fluid,  or  Mueller's  fluid,  give  less  satisfaction.  In  other  respects 
the  details  of  dehydration  and  embedding  are  the  same  as  those  used  in 
histological  studies,  except  that  it  is  desirable  that  the  tissues  should  be 
handled  rather  more  carefully  than  is  necessary  for  ordinary  patholog- 
ical work,  and  the  changes  from  the  weaker  to  the  stronger  alcohols 
should  be  made  less  abruptly.^ 

Embedding  in  paraffin  is  preferable  to  celloidin,  although  the  latter 
method  is  not  unsuccessful  if  carefully  carried  out.  The  chief  disadvan- 
tages of  celloidin  are  the  retention  of  color  by  the  celloidin  itself  and  the 
consequent  unclearness  of  differentiation.  It  is  also  easier  to  cut  thin 
sections  from  paraffin  blocks  than  from  those  prepared  with  celloidin. 

When  staining  tissue  sections  for  bacteria,  it  is  most  convenient 
to  carry  out  the  process  with  the  section  attached  to  a  slide.  For  cel- 
loidin sections  this  may  be  accomplished  by  means  of  ether  vapor.  For 
paraffin  sections  it  is  necessary  to  cover  the  slide  with  an  extremely  thin 
layer  of  a  filtered  mixture  of  equal  quantities  of  egg  albumin  and  glycerin, 
to  which  a  small  crystal  of  camphor  or  a  drop  or  two  of  carbolic  acid 
has  been  added.  The  sections  are  then  floated  upon  a  slide  so  prepared, 
and  set  away  in  the  thermostat  for  four  or  five  hours. 

Loepfler's  Method.^ — Stain  in  alcoholic  methylene-blue  solution 
five  to  fifteen  minutes,  or  in  Loeffler's  alkaline  methylene-blue  solution 
one  to  twenty-four  hours. 

Wash  in  one  to  one -thousand  acetic  acid  solution  for  about  ten 
seconds. 

Treat  with  absolute  alcohol  by  pouring  the  alcohol  over  the  prepara- 
tion for  ten  to  twenty  seconds. 

Clear  with  xylol. 

Mount  in  balsam. 

When  celloidin  sections  are  stained  in  this  way  ninety-five  per  cent 
alcohol  should  be  substituted  for  the  absolute.  A  number  of  other 
staining  solutions  may  be  used  in  the  same  way,  aqueous  fuchsin  or 
aqueous  gentian-violet  yielding  good  result. 


»  For  details  of  such  work  reference  should  be  had  to  the  standard  textbooks  on 
pathological  technique,  notably  the  very  excellent  one  of  Mallory  and  Wright. 
2  Loejffler,  Mitt.  a.  d.  kais.  Gesundheitsamt,  ii.  1884. 


MiCROSCOiPIC  STUDY  AND  STAINING  111 

NicoUe  advises  the  use  of  a  ten  per  cent  aqueous  solution  of  tannic 
acid  for  a  few  seconds  after  washing  with  the  acetic  acid. 

Sections  may  also  be  stained  by  placing  them  over  night  into  a 
dilute  Giemsa  solution  (one  drop  to  each  c.c.  of  distilled  water).  When 
so  stained  the  sections  must  not  be  run  through  the  weaker  alcohols 
but  must  be  rapidly  differentiated  in  absolute  alcohol. 

Method  of  Staining  Gram-Positive  Bacteria  in  Tissue  Sections. 
— Celloidin  Sections. — After  fixing  section  to  the  slide  by  pressure  with 
a  filter  paper  or  by  ether  vapor,  cover  with  anihn-water  gentian-violet 
five  minutes. 

Pour  off  excess  of  stain  and  cover  with  Gram's  iodin  solution  for 
two  minutes.  Decolorize  with  ninety-five  per  cent  alcohol  until  no 
more  color  comes  out. 

Stain  quickly  with  eosin-alcohol  (ninety-five  per  cent  alcohol  to 
which  enough  eosin  has  been  added  to  give  a  transparent  pink  color: 
about  1  :  15).  Clear  in  eosin-oil  of  origanum  (oil  of  origanum,  25  c.c. 
and  eosin  alcohol,  as  above,  about  3  c.c).    Blot  and  mount  in  balsam. 

Paraffin  Sections. — Stain  with  anilin-water  gentian-violet  five  to  ten 
minutes. 

Wash  in  water. 

Cover  with  Gram's  iodin  solution  one  minute. 

Wash  in  water. 

Decolorize  with  absolute  alcohol  until  no  more  color  comes  out. 

Clear  in  xylol. 

Mount  in  balsam. 

Gram-Weigert  Method.^ — (For  celloidin  sections.) — Stain  for  one-half 
hour  in  the  following  freshly  filtered  solution: 

Carmine * 3-5  grams. 

Saturated  aqueous  solution  of  lithium  carbonate 100  c.c. 

Dehydrate  in  ninety-five  per  cent  alcohol. 

Stick  section  to  slide  with  ether  vapor. 

Stain  in  anilin-water  gentian-violet  for  five  to  fifteen  minutes  (or 
in  a  saturated  solution  of  aqueous  crystal  violet  diluted  with  water  one 
to  ten,  five  to  fifteen  minutes) . 

Wash  in  physiological  salt  solution. 

Cover  with  Gram's  iodin  solution  one  to  two  minutes. 

Wash  in  water  and  blot. 

1  Wmgert,  Fortschr.  d.  Med.,  v,  1887. 


112  BIOLOGY  AND  TECHNIQUE 

Decolorize  with  anilin  oil  until  no  more  color  comes  off. 

This  both  decolorizes  and  dehydrates. 

Treat  with  xylol.     Mount  in  balsam. 

Method  of  Staining  for  Tubercle  Bacilli  in  Sections.^ — 
Paraffin  Sections. — Stain  in  carbol-fuchsin  solution  hot  for  five  minutes 
(or  better  cold,  for  twenty-four  hours) . 

Wash  in  water. 

Decolorize  and  counterstain  in  Gabbet's  methylene-blue  sulphuric 
acid  mixture  for  one  minute.  , 

Wash  in  water. 

Dehydrate  in  absolute  alcohol. 

Clear  in  xylol. 

Mount  in  balsam. 

Celloidin  Sections.'^ — Stain  lightly  in  alum  hematoxylin.  » 

Wash  in  water.  | 

Dehydrate  in  ninety-five  per  cent  alcohol.  I 

Attach  the  sHde  by  ether  vapor.  .  ? 

Stain  with  steaming  carbol-fuchsin  two  to  five  minutes.  \ 

Wash  in  water.  • 

Wash  with  Orth's  acid  alcohol  (alcohol  ninety  per  cent.,  99  c.c;  1 

cone.  HCl,  1  c.c.)  one-half  to  one  minute.  ' 

Wash  in  water  several  changes.  ' 

Treat  with  ninety-five  per  cent  alcohol  until  red  color  is  entirely 
gone.  ^  ^  ^  ^  I 

Blot  and  cover  with  xylol  until  clear.     Mount  in  balsam. 

Method  of  Staining  Actinomyces  in  Sections. ^Ma//or?/'s  Method^. 
— 1.  Stain  deeply  in  saturated  aqueous  eosin  ten  minutes, 

2.  Wash  in  water. 

3.  Anilin  gentian-violet  two  to  five  minutes. 

4.  Wash  in  normal  saline  solution. 

5.  Weigert's  iodin  solution  (iodin  1,  KI  2,  and  water  100  parts) 
one  minute. 

6.  Wash  in  water  and  blot. 

7.  Clear  in  anilin  oil. 

8.  Xylol  several  changes. 

9.  Mount  in  balsam. 


»  Mallory  and  Wright,  "  Pathol.  Tech.,"  p.  413. 

2  After  Mallory  and.  Wright. 

» Mallory  and  Wnght,  "  Pathol.  Tech.,"  1904 


CHAPTER  VII 

THE  PREPARATION  OF  CULTURE  MEDIA 

GENERAL   TECHNIQUE 

The  successful  cultivation  of  bacteria  upon  artificial  media  requires 
the  establishment  of  an  environment  which  shall  be  suitable  in  regard  to 
the  presence  of  assimilable  nutritive  material,  moisture,  and  osmotic 
relations.  These  requirements  are  fulfilled  in  the  composition  of  the 
nutrient  media  described  in  another  section,  media  which  are  to  some 
extent  varied  according  to  the  special  -requirements  of  the  bacteria 
which  are  to  be  cultivated.  If  cultivation,  furthermore,  is  to  have  any 
value  for  scientific  study  of  individual  species,  it  is  necessary  to  ob- 
tain these  species  free  from  other  varieties  of  microorganisms,  that  is, 
in  pure  culture,  and  to  protect  such  cultures  continuously  from  con- 
tamination with  the  other  innumerable  species  which  are  everywhere 
present. 

The  technique  which  is  employed  for  these  purposes  has  been  gradu- 
ally evolved  from  the  methods  originally  devised  by  Pasteur,  Koch. 
Cohn,  and  others. 

Bacterial  cultivation  is  carried  out  in  glassware  of  varied  construc- 
tion, the  forms  most  commonly  employed  being  test  tubes  of  various 
sizes,  Erlenmeyer  flasks,  the  common  Florence  flasks,  and  Petri  dishes. 
All  glassware,  of  course,  must  be  thoroughly  cleansed  before  being  used. 

Preparation  of  Glassware. — The  cleansing  of  glassware  may  be  ac- 
complished by  any  one  of  a  number  of  methods.  New  glassware  may 
be  immersed  in  a  one  per  cent  solution  of  hydrochloric  or  nitric  acid  in 
order  to  remove  the  free  alkali  which  is  occasionally  present  on  such  glass. 
It  is  then  transferred  to  a  one  per  cent  sodium  hydrate  solution  for  a 
few  hours,  and  following  this  is  washed  in  hot  running  water. 

In  the  case  of  old  glassware  which  has  contained  culture  media, 
sterilization  in  the  autoclave  is  first  carried  out,  then  the  glassware  is 
boiled  in  five  per  cent  soda  solution  or  in  soapsuds.  After  this,  thorough 
mechanical  cleansing  is  practiced,  and  the  glassware  may  be  treated  by^ 
acid  and  alkali  followed  by  running  water,  as  given  above.    These  last 

113 


114 


BIOLOGY  AND  TECHNIQUE 


steps,  however,  are  not  essential,  thorough  washing  in  hot  water  after 
the  soapsuds  or  soda  solution  being  usually  sufficient  to  yield  good 
results.  Other  workers  have  recommended  immersion  of  the  glassware 
after  mechanical  cleansing  in  five  per  cent  to  ten  per  cent  potassium 
bichromate  solution  in  twenty-five  per  cent  sulphuric  acid.  This  is 
followed  by  thorough  washing  in  hot  running  water,  and  drying. 

Clean  flasks  and  test  tubes  are  then  stoppered  with  cotton,  which  has 
been  found  to  be  a  convenient  and  efficient  seal  against  the  bacteria 
of  the  air,  catching  them  in  the  meshes  of  the  fibers  as  in  a  filter.  The 
technique  of  the  stoppering  or  plugging  of  glass  receptacles  is  important. 


Fig.  15. — Florence  Flask. 


Fig.  16. — Erlenmeyer  Flask. 


in  that,  when  poorly  plugged,  sterility  is  not  safeguarded,  and  the  pur- 
pose of  culture  study  is  defeated. 

In  almost  all  laboratories  in  this  country  non-absorbent  cotton  or 
"  cotton  batting  "  is  used  for  the  plug.  In  a  few  of  the  German  labora- 
tories the  absorbent  variety  is  employed.  The  disadvantages  of  the 
latter,  especially  in  the  case  of  fluid  media,  are  obvious.  The  plugs 
should  fit  snugly,  but  not  so  tightly  that  force  is  necessary  to  remove 
them.  Care  should  be  taken,  furthermore,  that  no  creases  are  left  be- 
tween the  surface  of  the  glass  and  the  periphery  of  the  plug;  for  these, 
if  present,  may  serve  as  channels  for  the  entrance  of  bacteria.  Fig. 
18,  accompanying,  will  illustrate  some  of  the  more  common  and  un- 
desirable defects  in  poorly  made  plugs.  The  plugging  itself  is  carried 
out  by  tearing  a  small  piece  of  cotton,  about  2  X  2  inches,  from  the  roll, 


THE  PREPARATION  OP  CULTURE  MEDIA 


115 


folding  over  one  of  its  corners,  and,  applying  the  smooth  end  of  a  "glass 
rod  to  the  folded  portion,  gently  pushing  it  into  the  mouth  of  the  tube. 
After  plugging  and  before  media  are  introduced  into  the  tubes  and 
flasks,  these  should  be  steriHzed.  This  is  best  done  in  one  of  the  "  hot- 
air  sterilizers"  (see  Fig.  8,  p.  69),  by  exposing  the  tubes  for  one  hour 
to  a  temperature  of  150°  C.  If  greater  speed  is  desired  exposure  to  180° 
to  190°  C.  for  half  an  hour  is  usually  safe.  If  by  mistake,  however,  the 
temperature  is  allowed  to  rise  above  200°  C,  a  browning  of  the  cotton 
plugs  occurs  and  the  glassware  is  apt  to  be  stained  by  the  burning  of 
the  fat  and  other  organic  material  derived  from  the  cotton.    Petri  dishes 


ce 


Fig.  17. — Petri  Dish. 


m 


after  cleansing  are  fitted  together  in  the  manner  shown  in  Fig.  17, 
and  are  sterilized  in  the  hot-air  chamber  at  150°  C.  for  one  hour. 

Glassware  so  prepared  is  ready  for  the  reception  of  media. 

Ingredients  of  Culture  Media. — The  food  requirements  of  bacteria 
have  been  discussed  in  another  section.  From  what  has  there  been 
said,  it  is  apparent  that  artificial  culture  media  must,  to  a  certain  extent, 
be  adjusted  to  the  peculiarities  of  individual  bacteria.  In  the  cases  of 
the  more  strictly  parasitic  microorganisms  growth  can  be  obtained  only 
by  the  most  rigid  observance  of  special  requirements.  For  the  large 
majority,  of  pathogenic  bacteria,  however,  routine  or  standard  media 
may  be  employed,  which,  while  slightly  more  favorable  for  one  species 
than  for  another,  are  sufficiently  general  in  their  composition  to  per- 
mit the  growth  of  all  but  the  most  fastidious  varieties. 

The  basis  of  many  of  our  common  media  is  formed  by  the  soluble 
constituents  of  meat.  These  substances  are  best  obtained  by  macerating 
500  grams  of  lean  beef  in  1,000  c.c.  of  distilled  water.    The  mixture  i^ 


116 


BIOLOGY  AND  TECHNIQUE 


K^ 


V.N. 


allowed  to  infuse  in  the  ice  chest  over  night,  and  then  strained  through 
cheese-cloth.  To  this  infusion  are  added  the  other  required  constituents 
m  the  manner  given  in  the  detailed  instructions  below.  The  soluble 
constituents  of  meat,  however,  may  also  be  procured  in  a  simpler  way 

by  the  use  of  the  commercial  meat  extracts, 
such  as  that  of  Liebig.  These  extracts  are 
dissolved  in  quantities  of  five  grams  to  the 
liter,  and  other  constituents  are  added  to 
this  nutrient  basis. 

Though  simpler  to  make,  the  meat-ex- 
tract media  are  less  favorable  for  the  culti- 
vation of  the  more  delicate  organisms  than 
are  the  media  made  directly  from  fresh  meat. 
Nevertheless,  they  suffice  for  the  cultivation 
of  the  large  majority  of  the  more  saprophytic 
pathogenic  microorganisms  and  hold  an  im- 
portant place  in  laboratory  technique. 

The  ingredients  and  methods  used  in  va- 
rious laboratories  in  the  preparation  of  such 
standard  media  should  be,  as  much  as  pos- 
sible, uniform,  in  order  that  confusion  in  re- 
sults may  be  avoided ;  for,  as  is  well  known, 
the  biological  characteristics  of  one  and  the 
same  bacterial  species  may  var}'  considerably 
if  grown  on  media  differing  in  their  compo- 
sition. 

A  committee  of  the  American  Public 
Health  Association,^  appointed  in  1897  for 
the  sake  of  standardizing  the  methods  of  preparation  of  media,  recom- 
mended that  the  following  rules  should  govern  the  choice  of  ingredients: 

1.  Distilled  water  should  be  used  in  all  cases. 

2.  The  meat  used  should  be  fresh,  lean  beef  (when  veal  or  chicken 
is  substituted  the  change  should  be  stated). 

3.  The  pepton  used  should  be  Witte's  pepton,  dry,  made  from  meat. 

4.  Only  C.  P.  NaCl  should  be  used. 

5.  For  alkalinizing  C.  P.  sodium  hydrate  should  be  used  in  normal 
solutions. 


b 


Fig.  18.— Test  Tube  (a) 
incorrectly  stoppered;  (b) 
correctly  stoppered. 


^Rep.   Com.  of    Amer.  Bact.  to  Com.  of    Amer.  Pub.  Health  Assn.  Meeting, 
Philadelphia,  Sept.,  1897. 


THE  PREPARATION  OF  CULTURE  MEDIA 


117 


6.  For  acidification  C.  P.  hydrochloric  acid  in  normal  solution  should 
be  used. 

7.  When  glycerin  is  used,  this  should  be  of  the  redistilled  variety. 

8.  The  agar-agar  employed  should  be  of  the  finest  grade  of  commer- 
cial thread  agar. 

9.  The  gelatin  should  be  the  commercial  sheet  gelatin  washed  as 
free  as  possible  of  acid  and  impurities. 

10.  Chemicals  and  carbohydrates   which   are   used   should   be   as 
nearly  chemically  pure  as  possible. 

Titration  of  Media. — Next  in 
importance  to  the  actual  composi- 
tion of  media  is  the  adjustment  of 
their  reaction.  Bacteria  are  highly 
susceptible  to  variations  in  the 
acidity  and  alkalinity  of  media, 
excessive  degress  of  either  may 
completely  inhibit  development  or 
moderate  variations  may  lead  to 
marked  modifications  of  cultural 
characteristics.  It  is  necessary, 
therefore,  to  adjust  the  reaction 
both  for  the  sake  of  favoring 
growth  and  in  order  to  insure  uni- 
formity of  growth  characters.  This 
is  accomplished  by  titration  which 
is  best  carried  out  according  to  the 
recommendations  of  the  committee 
mentioned  above. 

The  color  indicator  employed  for* 
the  titration  is  a  five-tenths  per  cent 
solution  of  phenolphthalein  in  fifty 
per  cent  alcohol.  The  chief  advan- 
tage of  this  indicator  over  others  is 
due  to  the  fact  that  it  indicates  the 
presence  of  organic  acid  and  acid 
compounds    in   its    reaction.     For 

actual  titration  ^  (^  normal)  solutions  of  sodium  hydrate  or  of  hy- 
drochloric acid  are  used.  Since  media  in  the  process  of  preparation 
are  usually  acid,  the  NaOH  solution  is  the  one  most  frequently  needed. 
F**^  c.c.  of  the  medium  to  be  tested  is  measured  accurately  in  a  care- 


FiG.  19. — Burette  for  Titrating 
Media. 


118 


BIOLOGY  AND  TECHNIQUE 


fully  washed  pipette  and  transferred  into  a  porcelain  evaporating  dish, 
to  this  are  added  45  c.c.  of  distilled  water.  The  mixture  is  thoroughly 
boiled  for  three  minutes  oVer  a  free  flame.  The  boiling  drives  off  CO2, 
giving  the  true  neutral  point,  and  approximates  the  conditions  prevaiUrig 

during  the  further  sterilization  of 
the  medium  from  which  the  5  c.c. 
have  been  taken.  After  boiling,  1 
c.c.  of  the  phenolphthalein  is  added. 
If  the  medium  is  acid,  no  color  is 
present;  if  alkaline,  a  pink  or  red 
color  appears.  The  -^  alkali  or 
acid  solution  is  allowed  to  drop 
into  the  dish  from  a  graduated 
burette.  When  the  neutral  point 
is  approached  in  an  acid  solution, 
each  drop  of  sodium  hydrate  added 
brings  forth  at  first  a  deep  red, 
which,  however,  upon  slight  stir- 
ring with  a  clean  rod,  completely 
disappears.^  The  end  reaction  is 
reached  when  a  faint  but  clear  arid 
distinct  pink  color  remains  in  the 
fluid  after  stirring. 

When  titrating  alkaline  media^ 
the  addition  of  the  phenolphthalein 
produces  a  red  color  in  the  hot 
medium  which  gradtially  fades  upon 
the  addition  of  |^ "  HCl,  bdcbming 
colorless  at  the  end  point  of  titration.  Titration  should  be  done 
quickly  and  in  a  hot  solution.  From  the  result  of  the  titration  the 
computation  for  the  neutralization  of  the  entire  bulk  of  the  rriedium 
can  be  made  by  a  simple  arithmetical  process  as  illustrated  in  the 
following  example: 

Let  us  suppose  that  we  have  used: 


Fig.  20. — Tubing 'Media. 


2.5  c.c.  of  ^  NaOH  to  neutralize 

N 
T 
N 


then  2.5  c.c.  of 
and    25  c.c.  of 


NaOH  will  neutralize 


5  c.c.  of  the  medium, 
100  c.c.      " 


NaOH  will  neutralize  1,000  c.c,  or  one  liter. 


See  standard  textbooks  on  volumetric  analysis. 


THE  PREPARATION  OF  CULTURE  MEDIA 


119 


/:?M; 


'f'o'fy"  ."> . 


m 


The  adjustment  of  the  reaction  of  media  is  largely  determined  by  the 
particular  uses  for  which  the  media  are  designed.  For  examinations  iii 
the  practice  of  sanitation,  such  as  analyses  of  water,  ice,  and  milk,  etc., 
the  American  Public  Health  Association  recommends  a  standard  reac- 
tion of  +  1  per  cent  (the  plus  sign  is  used  to  indicate  acidity,  the  minus 
alkalinity;  +  1  per  cent  is  the  expression  used  to  indicate  that  one  per 
per  cent  of  ^  sodium  hydrate  solution  would  be  required  to  neutralize 
the  medium  or  10  c.c.  to  the  Uter).  For  general  work  with  pathogenic 
bacteria,  the  most  favorable  reaction  for  routine  media  is  slight  alka- 
Hnity,  neutrality,  or  an  acidity  not  exceeding  +  1  per  cent. 

Methods   of    Clearing    Media. — Clearing  with  Eggs. — When  culture 
media  are  prepared  from  substances  containing  no  coagulable  proteid, 
it  is  often  necessary,  for  purposes  of  clearing,  to  add  the  whites  of  eggs, 
and  then  to  heat  for  forty-five  min- 
utes in  the  Arnold   sterilizer.      In 
the  following  detailed  descriptions, 
the  direction  "clear  with  egg"  has 
been  given  whenever  such  a  step  is 
deemed  necessary.    The  exact  tech- 
nique  of   such    a   procedure    is  as 
follows : 

In  a  small  pot  or  pan,  the 
whites  of  several  eggs  (one  or  two 
eggs  to  each  liter  of  medium)  are 
beaten  up  thoroughly  with  a  little 
water  (20  c.c).  This  egg  white  is 
then  poured  into  the  medium, 
which,  if  hot,  as  in  the  case  of 
melted  agar  or  gelatin,  must  first 
be  cooled  to  about  50°  to  55°  C. 
The  mixture  is  then  thoroughly 
shaken  and  steamed  in  the  Arnold 
sterilizer  for  thirty  minutes.  At 
the  end  of  this  time  the  flask  con- 
taining the  medium  is  removed  from 
the  sterilizer  and  thoroughly  shaken 

so  as  completely  to  break  up  the  coagulum  which  has  formed.  It  ia 
then  replaced  and  allowed  to  steam  for  another  fifteen  minutes.  At 
the  end  of  this  time  the  medium  between  the  coagula  should  be  clear. 
It  is  now.  ready  for  filtration  through  cotton. 


/■^A 


a  be 

Fig.  21. — Media  in  Tubes:  a,  broth; 
b,  agar  slant;  c,  potato. 


120 


BIOLOGY  AND  TECHNIQUE 


Filtering  Media  through  Cotton. — ^The  filtration  of  media  after 
clearing,  either  by  the  addition  of  eggs  or  by  the  coagulation  of  the  pro- 
teids  originally  contained  in  it,  is  best  done  through  absorbent  cotton. 
A  small  spiral,  improvised  of  copper  wire,  is  placed  as  a  support  in  the 
bottom  of  a  large  glass  funnel.    A  square  piece  of  absorbent  cotton  ie 


Fig.  22. — Berkefeld  Belter. 


then  split  horizontav/ giving  two  squares  of  equal  size.  Ragged  edges 
and  incisures  shou'd  be  avoided.  These  two  layers  of  cotton  are  then 
placed  in  the  funnel,  one  piece  above  the  other  in  such  a  way  that  the 
direction  of  the  fibers  of  the  two  layers  is  at  right  angles  one  td  the  other. 
They  are  then  gently  depressed  into  the  filter  with  the  closed  fist.    Th*^ 


THE  PREPARATION  OF  CULTURE  MEDIA 


121 


edges  of  the  cotton  are  made  to  adhere  to  the  sides  of  the  funnel  by 
allowing  a  thin  stream  of  tap  water  to  run  over  them,  while  smoothing 
them  against  the  glass  with  the  hand. 

The  medium,  when  poured  into  such  a  filter,  should  be  poured  along 
a  glass  rod  at  first,  to  avoid  running  down  the  sides  or  bursting  the  filter. 
After  filtration  has  begun,  the  filter  should 
be  kept  as  full  as  possible.  The  first  liter 
or  so  which  comes  through  may  not  be  clear, 
but  the  filter  gains  in  efficiency  as  the  coag- 
ulum  settles  into  the  fibers  of  the  cotton, 
and  the  first  yield  may  be  sent  through  a 
second  time.  Filtration  of  agar  or  gelatin  is 
best  done  in  a  warm  room  with  windows 
and  doors  closed,  and  the  filter  covered  with 
a  fid,  to  avoid  too  rapid  cooling.  The  funnel 
and  filter  should  be  warmed  just  before  use. 

Filtering  through  Paper. — Many  media 
may  be  efficiently  cleared  by  filtration 
through  close  filter  paper  without  the  aid  of 
coagula. 

The  Tubing  of  Media. — Most  of  the  media 
described  in  the  foregoing  section  are  used 
in  test  tubes.  In  order  to  fill  these  tubes, 
the  media  are  best  poured  into  a  large  glass 
funnel  to  which  a  glass  discharging  tube  has 
been  fitted  by  means  of  a  short  piece  of 
rubber  tubing  (see  Fig.  20) .  Upon  this  is 
placed  a  thumb  cock.  The  plug  is  then  re- 
moved from  the  test  tube  by  catching  it  be- 
tween the  small  and  ring  fingers  of  the  right 
hand  and  the  glass  outlet  is  thrust  deeply 
into  the  test  tube,  in  order  to  prevent  the 
medium  from  touching  the  upper  portion 
of  the  test  tube  where  the  cotton  plug 
will  be  lodged.     About  7  to  8  c.c.  is  put  in  each  test  tube. 

Sterilization  of  Media. — By  Heat. — Media  which  contain  neither 
sugars,  gelatin,  glycerin,  nor  animal  serum  may  be  sterilized  in  the  auto- 
clave at  fifteen  pounds  pressure  for  fifteen  minutes  to  half  an  hour. 
Media  which  contain  these  or  other  substances  subject  to  injury  from 
the  high  temperature,  must  be  sterilized  by  the  fractional  method, 


Fia.  23. — Berkeffld 
Filter. 


122 


BIOLOGY  AND  TECHNIQUE 


i.e.,  by  twenty  minutes'  exposure  in  the  live  steam  sterilizer  (Arnold, 
Fig.  9,  p.  70)  on  each  of  three  consecutive  days.  During  the  intervals 
between  sterilizations,  they  should  be  kept  at  room  temperature  or  in  the 
incubator,  to  permit  the  germination  of  spores  which  may  be  present. 
Media  containing  animal  serum  or  other  albuminous  solutions  which 
are  to  be  sterilized  without  coagulation,  may  be  sterilized  in  wate 
baths,  or  in  hot-air  chambers  (Fig.  10,  p.  71),  at  temperatures  varying 


Fig.  24. — Reichel  Filter. 


from  60°  to  70°  C,  by  the  fractional  method.    In  such  cases  five  or 
six  exposures  of  one  hour  on  succeeding  days  should  be  employed. 

By  Filtration. — It  is  often  desirable  in  bacteriological  work  to  free 
fluid  from  bacteria.  This  is  frequently  necessary  for  the  sterilization 
of  blood-serum  or  exudate  fluids,  or  for  obtaining  toxins  free  from  bac- 
teria. For  these  purposes  a  large  variety  of  filters  are  in  use.  Those 
most  commonly  employed  are  of  the  Chamberland^  or  Berkefeld  type, 
which  consist  of  hollow  candles  made  of  unglazed  porcelain  or  dia- 
tomaceous  earth.  Both  these  types  are  made  in  various  grades  of  fine- 
ness, upon  which  depend  both  the  speed  of  filtration  and  the  efficiency. 
They  are  made  in  various  forms  and  models,  some  of  which  are  shown 

1  Pasteur  and  Chamberland,  Compt.  rend,  de  Tacad.  des  sci.,  1884. 


THE   PREPARATION   OF  CULTURE   MEDIA 


1^ 


in  the  accompanying  figures.  In  most  of  the  methods  of  filtration 
commonly  employed  the  fluid  which  is  to  be  filtered  is  sucked  through 
the  walls  of  the  filter,  either  by  a  hand  suction-pump  or  by  some  form 
of  vacuum-pump  attached  to  an  ordinary  water-tap. 

The  hollow  candle-filter  may  either  be  firmly  fitted  into  a  cylin- 
drical glass  chimney  and  surrounded  by  the 
fluid  which  is  to  be  filtered,  or  else  the  candle 
may  be  connected  to  the  collecting  flask 
with  sterile  rubber  tubing  and  suspended 
freely  in  the  fluid.  Perfect  filters  of  these 
types  will  hold  back  any  of  the  bacteria 
known  to  us  at  present. 

Filters  before  use  must  be  sterilized. 
The  candles  themselves  are  subjected  to 
150°  C.  in  the  hot-air  sterilizer  for  one  hour. 
The  glassware  and  washers  necessary  for 
setting  up  the  apparatus  may  be  sterilized 
by  boiling.  In  order  that  filters  may  be  re- 
peatedly used  with  good  result,  it  is  neces- 
sary that  they  should  be  carefully  cleaned 
from  time  to  time.  This  is  best  done  in  the 
following  way: 

Filters  through  which  fluids  from  living 
cultures  have  passed  are  first  sterilized  in 
the  Arnold  steam  sterilizer.  Their  exterior 
is  then  carefully  cleaned  with  a  fine  brush. 
Following  this  a  five-tenths  per  cent  solu- 
tion of  potassium  permanganate  is  passed 
through  them  and  this  again  removed  by 
sucking  through  a  five  per  cent  solution  of 
bisulphite  of  soda.  This  last  is  washed  out 
by  sending  a  considerable  quantity  of  dis- 
tilled water  through  the  filter,  which  is  then  dried  and  sterilized  by 
heat. 

The  suction  necessary  for  filtration  through  these  filters  is  usually 
applied  by  means  of  the  ordinary  suction-pump  attached  to  a  running 
faucet. 

Slanting  of  Media. — Solid  media  which  are  to  be  used  in  slanted  form 
in  test  tubes  should  be  inclined  on  a  ledge  (easily  improvised  of  glass 
tubing)  at  the  proper  slant,  after  the  last  sterilization.   Agar,  the  medium 


Fig.  25. — Kitasato  Filter. 


124 


felOLOGY  ANt)  TECHNIQUE 


most  frequently  employed  in  this  way,  should  be  left  in  this  position 
for  two  or  three  days.     (See  Fig.  21,  b.) 

ACTUAL    STEPS    IN    THE    PREPARATION    OF    NUTRIENT    MEDIA 


Broth. — Meat  Extract  Broth. — 1.  To  1,000  c.c.  of  distilled  or  clear 
tap  water  add  5  gms.  Liebig's  meat  extract,  10  gms.  Witte's  pepton, 

and  5  gms.  common  salt  (NaCl). 

2.  Weigh  solution  with  containing 
vessel  (any  suitable  agate-ware  vessel 
or  glass  flask  will  do). 

3.  Heat  over  free  flame  until  thor- 
oughly dissolved,  stirring  constantly. 

4.  Weigh  again  and  make  up  loss 
by  evaporation. 

5.  Determine  volume. 

6.  Titrate  and  adjust  to  required 
reaction,  heating  over  free  flame  for 
five  minutes. 

7.  Filter  through  paper  until  clear. 

8.  Sterihze. 
If  medium  can  not  be  cleared  by 

filtering  through  paper,  clearing  by 
white  of  egg  msLj  be  resorted  to  and 
the  medium  filtered  through  cotton. 

Meat  Infusion  Broth. — 1.  Infuse 
500  gms.^  of  lean  meat,  twelve  to 
twenty-four  hours,  with  1,000  c.c.  of 
distilled  water  in  refrigerator. 

2.  Strain  through  wet  cotton  flan- 
nel or  wet  cheese-cloth  and  make  up 
volume  to  1,000  c.c. 

3.  Add  5  gms.  common  salt  and 
10  gms.  Witte's  pepton. 

4.  Weigh  with  containing  vessel. 

5.  Warm  over  flame  or  water  bath,  stirring  until  pepton  is  dissolved, 
not  allowing  temperature  to  rise  above  50°  C. 

6.  Determine  volume. 


Fig.    26. — ^Maassen    Filter,    for 
SMALL  Quantities  of  Fluid. 


Roughly,  1  pound  (1^  lb.). 


THE   PREPARATION   OF   CULTURE    MEDIA  125 

7.  Titrate  and  adjust  reaction  to  neutral. 

8.  Heat  in  Arnold  steriliser  for  thirty  minutes;  shake  or  stir  well 
and  heat  again  for  fifteen  minutes. 

9.  Determine  weight  and  restore  loss  by  evaporation. 

10.  Determine  volume,  titrate,  and  adjust  reaction  to  desired  point 
(usually  one  per  cent  acid). 

11.  Heat  again  for  five  minutes  if  adjustment  of  reaction  has  been 
necessary. 

12.  Filter  through  absorbent  cotton,  passing  the  filtrate  through 
the  same  filter  until  clear. 

13.  Titrate  and  record  the  final  reaction. 

Place  in  cotton-plugged  sterile  flasks  or  plugged  sterile  test  tubes, 
and  sterilize  for  thirty  minutes  in  the  Arnold  sterilizer  on  three  suc- 
cessive days,  leaving  at  room  temperature  in  the  intervals. 

Sugar-Free  Broth. — 1.  Make  1  liter  of  meat  infusion  broth,  following 
steps  1,  2,  3,  4,  5,  6,  7,  and  8\*  then  filter  through  thin  cotton  filter  to 
remove  gross  particles — total' clearing  is  not  necessary. 

2.  Put  the  broth  in  a  flask  and  cool.  Then  add  10  c.c.  of  a  twenty- 
four-hour  broth  culture  of  B.  coli  communis. 

3.  Place  the  flask,  stoppered  with  cotton,  in  the  incubator  at  37°  C. 
for  eighteen  hours.  (The  bacteria  will  ferment  and  thus  destroy  any 
sugar  [monosaccharid]  which  may  be  present  in  the  broth,  and  thus 
render  the  broth  sugar-free  and  acid.) 

4.  Heat  thoroughly  to  kiU  the  bacteria. 

5.  Determine  weight  and  bring  to  1,015  gms.  Then  determine 
volume  and  titrate,  and  adjust  to  neutral.    Heat  thoroughly  again. 

6.  Filter  through  filter  paper  until  clear. 

7.  The  pure  sugars,  dextrose,  lactose,  saccharose,  etc.,  are  then  added 
to  separate  portions  (250  c.c.)  of  the  broth  in  the  proportion  of  one 
per  cent. 

8.  When  the  sugars  are  dissolved,  tube  the  broth  immediately  in 
fermentation  tubes,  and  sterilize  by  discontinuous  sterilization,  never 
heating  over  twenty  minutes  at  a  time,  as  heat  tends  to  destroy  or 
change  the  sugars. 

Glycerin  Broth. — To  ordinary,  slightly  acid  or  neutral  meat  in- 
fusion broth,  add  six  per  cent  of  C.  P.  glycerin.  Sterilize  by  frac- 
tional method. 


*  These  steps  refer  to  the  regular  directions  for  making  infusion  broth.     Odlq 
liter  of  previously  made  ijof usion  broth  may  be  used  instead. 


'™ 


126  BIOLOGY  AND  TECHNIQUE 

Calcium  Carbonate  Broth. — This  medium  is  designed  for  obtaining 
mass  cultures  of  pneumococcus  or  strepttococcus  for  purposes  of  im- 
munization or  agglutination. 

To  100  c.c.  of  meat  infusion  broth  in  small  flasks,  add  one  per  cent 
of  powdered  calcium  carbonate,  and  one  per  cent  of  glucose.  It  is  a 
wise  precaution  to  sterilize  the  dried  calcium  carbonate  in  the  hot-air 
chamber  before  using.  Small  pieces  of  marble  may  be  used  as  sug- 
gested by  Bolduan. 

Pepton-Salt Solution  (Dunham's  solution): 

1.  Distilled  water 1,000  c.c. 

Pepton  (Witte) 10  gms. 

NaCl 5     " 

2.  Heat  until  ingredients  are  thoroughly  dissolved. 

3.  Filter  through  filter  paper  until  perfectly  clear. 

4.  Tube  twenty-five  tubes,  and  store  remainder  in  250  c.c.  flasks. 
Sterilize  by  discontinuous  method. 

Nitrate  Solution. — 

1.  Distilled  water 1,000      c.c. 

Pepton 10     g;m.<. 

Potassium  nitrate 0.2   " 

2.  Heat  until  ingredients  are  thoroughly  dissolved. 

3.  Filter  through  filter  paper  until  perfectly  clear. 

4.  Tube  twenty-five  tubes,  and  store  remainder  in  250  c.c.  flasks. 
Sterilize  by  discontinuous  sterilization. 
UscMnsky^sProteid-Free  Medium} — To  one  liter  of  distilled  water  add: 

Asparagin 3.4  grams. 

Ammonium  lactate , 10  " 

Sodium  chloride 5     .     " 

Magnesium  sulphate 0.2      " 

Calcium  chloride . 0.1       " 

Potassium  phosphate 1.0      " 

When  these  substances  are  thoroughly  dissolved,  add  40  c.c.  of  glycerin 
Tube  and  sterilize. 

Gelatin. — Meat-Extract  Gelatin.^1.  To  1,000  c.c.  of  distilled  water 
add  Liebig's  extract  5  gms.,  pepton  10  gms.,  NaCl  5  gms.,  and  120 
gms.  of  the  finest  French  sheet  gelatin.^ 

1  Uschinsky,  Cent.  f.  Bakt.,  1,  xiv,  1*893. 

2  The  acidity  and  consistence  of  the  different  commercial  gelatins  vary  con- 
siderably and  care  should  be  taken  in  selecting  a  uniform  and  suitable  brand,  such  as 
Hesterberg's  gold  label  gelatin.  It  is  advisable,  when  working  during  the  summer 
or  in  hot  climates,  to  add  130  instead  of  120  grams. 


THE    PREPARATION   OF   CULTURE    MEDIA  127 

2.  Weigh  with  containing  vessel. 

3.  Dissolve  by  warming. 

4.  Adjust  weight,  determine  volume,  titrate,  and  adjust  reaction. 

5.  Cool  to  60°  C,  add  whites  of  two  eggs,  and  stir  thoroughly. 

6.  Heat  for  thirty  minutes,  stir  thoroughly,  and  heat  for  fifteen 
minutes. 

7.  Adjust  weight. 

8.  Filter  through  cotton. 

9.  SteriHze. 

Meat-Infusion  Gelatin. — 1.  Infuse  500  gms.  lean  meat  twelve  to 
twenty-four  hours  with  1,000  c.c.  of  distilled  water  in  refrigerator, 

2.  Strain  through  wet  cotton  flannel  or  wet  cheese-cloth  and  make 
up  volume  to  1,000  c.c. 

3.  Add  5  gms.  common  salt,  10  gms.  Witters  pepton,  and  120  gms. 
of  the  finest  French  sheet  gelatin. 

4.  Weigh  with  containing  vessel. 

5.  Warm  over  flame  or  water  bath,  stirring  till  pepton  and  gelatin 
are  dissolved  and  not  allowing  temperature  to  rise  above  50°  C. 

6.  Determine  volume. 

7.  Titrate  and  adjust  reaction  to  neutral. 

8.  Heat  in  Arnold  sterilizer  for  thirty  minutes;  shake  or  stir  well 
and  heat  again  for  fifteen  minutes. 

9.  Determine  weight  and  restore  loss  by  evaporation. 

10.  Determine  volume,  titrate,  and  adjust  reaction  to  desired  point, 
if  necessary  (one  per  cent  acid). 

11.  Heat  five  minutes  over  free  flame,  constantly  stirring,  if  ad- 
justment of  reaction  has  been  necessary. 

12.  Filter-  through  absorbent  cotton,  passing  the  filtrate  through 
the  same  filter  until  clear. 

13.  Titrate  and  record  the  final  reaction. 

Place  gelatin  in  cotton-plugged  sterile  250  c.c.  flasks  or  about  8  c.c. 
in  plugged  sterile  test  tubes  and  sterilize  for  thirty  minutes  in  the  Arnold 
sterilizer  on  three  successive  days,  leaving  at  room  temperature  in  the  in- 
tervals. Never  heat  the  gelatin  for  longer  than  is  necessary  to  comply 
with  directions,  or  it  may  not  be  solid  enough  for  use.  With  some 
brands  of  gelatin  it  may  be  necessary  to  add  thirteen  per  cent  in  order 
to  obtain  sufficient  stiffness. 

Agar. — Meat-Extract   Agar. — 1.  To  1,000  c.c.  of  distilled  water  (or 
tap  water)  add  15  gms.  of  thread  agar,  10  gms.  of  Witte's  pepton,  and 
5  gms.  of  Liebig's  meat  extract,  and  5  gms.  of  common  salt. 
10 


128  BIOLOGY  AND  TECHNIQUE 

2.  Weigh  with  containing  vessel. 

3.  Heat  over  free  flame  until  agar  is  dissolved,  thirty  to  forty-five 
minutes.  (Great  care  should  be  exercised  in  determining  that  agar  is 
completely  in  solution.) 

4.  Determine  weight  and  make  up  loss  by  evaporation. 

5.  Determine  volume,  titrate,  and  adjust  to  desired  reaction. 

6.  Cool  to  60°  C. 

7.  Add  whites  of  two  eggs  and  stir  thoroughly. 

8.  Heat  in  Arnold  sterilizer  thirty  minutes,  stir,  and  reheat  fifteen 
minutes. 

9.  Weigh  and  make  up  loss  by  evaporation. 

10.  Determine  volume,  titrate,  and  correct  reaction  if  necessary.* 

11.  Heat  for  five  minutes,  if  reaction  is  corrected. 

12.  Filter  through  cotton,  tube,  and  sterilize. 

Meat-Infusion  Agar? — (A)  1.  Infuse  500  gms.  lean  meat  twelve  to 
twenty-four  hours  in  500  c.c.  of  distilled  water  in  refrigerator. 

2.  Strain  through  wet  cotton  flannel  or  wet  cheese-cloth,  and  make 
up  voiiime  to  500  c.c. 

3.  Add  10  gms.  of  Witte's  pepton  and  5  gms.  of  common  salt. 

4.  Weigh  solution  and  containing  vessel. 

5.  Warm  over  free  flame  or  water  bath  till  pepton  and  salt  are  dis- 
solved, not  allowing  temperature  to  rise  above  50°  C. 

6.  Determine  volume,  titrate,  and  neutralize. 

(B)  7.  Add  15  gms.  of  thread  agar  to  600  c.c.  of  distilled  water  and 
boil  over  free  flame  for  thirty  to  forty-five  minutes,  watching  and  stirring 
constantly,  or  preferably  heat  in  autoclave  till  agar  is  completely  dis- 
solved. This  will  lose  weight  by  evaporation ;  final  weight  should  be 
515  gms. 

8.  Cool  this  to  about  60°  C. 

(C)  9.  Then  to  the  solution  A  of  meat  infusion  (at  50°  C.)  add  the 
solution  B  of  agar  (at  60°  C). 

10.  Determine  volume,  titrate,  and  adjust  reaction  to  plus  one  per 
cent  acid  or  any  desired  reaction. 

11.  Heat  for  thirty  minutes  in  Arnold  sterilizer  or  autoclave. 
Shake  or  stir  thoroughly,  and  heat  fifteen  minutes  more.  Adjust 
weight  by  adding  water. 

*  While  titrating,  care  should  be  taken  that  medium  does  not  solidify  along  sides 
of  vessel.    Agar  may  be  made  more  quickly  and  successfully  in  autoclave. 

2  Glycerin  agar  is  made  by  adding  6  per  cent  of  C.  P.  glycerin  to  meat-extract 
or  meat-infusion  agar. 


THE  PREPARATION  OF  CULTURE  MEDIA  129 

13.  Filter  through  absorbent  cotton,  passing  the  filtrate  through  the 
same  filter  until  clear. 

14.  Titrate  and  record  final  reaction. 

Place  agar  in  cotton-plugged  sterile  flasks  or  plugged  sterile  test 
tubes  and  sterilize  for  thirty  minutes  on  three  successive  days. 

Lactose-Litmus-Agar  (Wurtz). — 1.  Put  1,500  c.c.  distilled  water  in 
previously  weighed  agate-ware  vessel. 

2.  Add  15  gms.  thread  agar  and  boil  over  free  flame  for  thirty  to 
forty-five  minutes,  watching  and  stirring  constantly  till  the  agar  is 
completely  dissolved. 

3.  Add  5  gms.  Liebig's  extract  of  meat,  5  gms.  NaCl,  10  gms.  Witte's 
pepton,  and  dissolve  completely. 

4.  Restore  loss  by  evaporation  to  1,035  gms. 

5.  Determine  volume,  titrate,  and  adjust  reaction  to  one  per  cent 
acid. 

6.  Place  in  a  flask  and  cool  to  60°  C. 

7.  Add  the  whites  of  two  eggs  beaten  up  in  50  c.c.  of  water  and  mix 
thoroughly. 

8.  Heat  for  thirty  minutes  in  Arnold  sterilizer,  shake  thoroughly, 
and  heat  again  for  fifteen  minutes. 

9.  Adjust  weight. 

10.  Filter  through  absorbent  cotton  to  clear. 

11.  Add  two  per  cent  pure  lactose  (milk  sugar).* 

12.  Add  enough  pure  five  per  cent  litmus  solution  ^  to  bring  to 
purple  color  when  cold. 

13.  Tube  and  sterilize. 

WelcNs  Modification  of  Guamieri's  Medium.^ — This  medium  is  made 
on  a  meat-infusion  basis,  according  to  the  directions  given  for  the  prep- 
aration of  meat-infusion  agar.  It  contains  5  grams  of  agar,  80  grams  of 
gelatin,  5  grams  of  NaCl,  and  10  grams  of  pepton  to  one  liter.    It  should 


*  Add  lactose  and  litmus  to  250  c.c.  for  25  tubes;  keep  the  remainder,  with- 
out lactose,  stored  in  small  sterile  flasks  for  further  use. 

2  The  litmus  solutions  used  in  the  preparation  of  media  are  best  made  up  as  fol- 
lows: Litmus  in  substance — Merck's  purified,  or  Kaulbaum's — is  dissolved  in  water 
to  the  extent  of  5  per  cent.  The  solution  is  made  by  heating  in  an  Arnold  sterilizer 
for  about  one  to  two  hours,  shaking  occasionally.  The  solution  is  then  filtered  through 
paper  and  sterilized.    It  should  be  kept  sterile,  as  molds  will  grow  in  it  otherwise, 

A  standard  litmus  solution,  which  is  marketed  for  laboratory  purposes,  known 
as  "Kubel  and  Tiemann's"  solution,  may  be  used. 

'  Welch,  Bull.  Johns  Hopkins  Hosp. 


130  BIOLOGY  AND  TECHNIQUE 

be  adjusted  to  a  neutral  reaction.    It  is  used  for  stab  cultures  and  is 
designed  chiefly  for  pneumococcus  cultivation  and  storage. 

Dorsett  Egg  Medium — This  medium  is  chiefly  useful  for  the  cul- 
tivation of  tubercle  bacilli. 

1.  Carefully  break  eggs  and  drop  the  contents  into  a  wide-mouthed 
flask.  Break  up  the  yolk  with  a  sterile  platinum  wire,  and  shake  up 
the  flask  until  the  whites  and  yolks  are  thoroughly  mixed. 

2.  Add  25  c.c.  of  distilled  water  to  every  four  eggs;  strain  through 
sterile  cloth. 

3.  Pour  10  c.c.  each  into  sterile  test  tubes  and  slant  in  an  inspissator 
and  expose  to  73°  C.  for  four  to  five  hours  on  two  days. 

4.  On  the  third  day,  raise  the  temperature  to  76°  C. 

5.  The  sterilization  may  be  finished  by  a  single  exposure  to  100° 
C.  in  the  Arnold  sterilizer  for  fifteen  minutes.  Before  inoculation,  add 
two  or  three  drops  of  sterile  water  to  each  tube. 

For  a  description  of  Petroff's  medium  for  the  isolation  of  tubercle 
bacilli,  see  page  484. 

Potato  Media. — Large  potatoes  are  selected,  washed  in  hot  water, 
and  scrubbed  with  a  brush.  They  are  peeled,  considerably  more  than 
the  cuticle  being  removed  The  peeled  potatoes  are  washed  in  running 
water,  following  which  cylindrical  pieces  are  removed  with  a  large 
apple  corer.    The  cylinders  are  cut  into  wedges. 

Since  the  reaction  of  the  potato  is  normally  acid,  this  should  be  cor- 
rected by  washing  the  pieces  in  running  water  over  night,  or,  better, 
by  immersing  them  in  a  one  per  cent  solution  of  sodium  carbonate  for  half 
an  hour. 

The  pieces  are  then  inserted  into  the  large  variety  of  test  tubes 
known  as  "potato  tubes."  (See  Fig.  21,  c.)  In  the  bottom  of  the 
tubes  a  small  amount  of  water  (about  1  c.c.)  or  a  small  quantity  of 
moist  absorbent  cotton  should  be  placed  in  order  to  retard  drying  out 
of  the  potato.  The  tubes  are  sterilized  by  fractional  sterilization, 
twenty  minutes  to  half  an  hour  in  the  Arnold  sterilizer  on  three  suc- 
cessive days. 

Glycerin  Potato. — In  preparing  glycerin  potato  the  potato  wedges 
are  treated  as  above,  and  are  then  soaked  in  a  ten  to  twenty-five  per 
cent  aqueous  glycerin  solution  for  one  to  three  hours.  A  small  quantity 
of  a  ten  per  cent  glycerin  solution  should  be  left  in  the  tubes.  In  steril- 
izing these  tubes,  thirty  minutes  a  day  in  the  Arnold  after  heating  of 
the  sterilizer  will  sterilize  without  altering  the  glycerin. 

Milk  Media. — Fresh  milk  is  procured  and  is  heated  in  a  flask  for 


THE  PREPARATION  OF  CULTURE  MEDIA         131 

fifteen  minutes  in  an  Arnold  sterilizer.  It  is  then  set  away  in  the  ice 
chest  for  about  twelve  hours  in  order*  to  allow  the  cream  to  rise.  Milk 
and  cream  are  then  separated  by  siphoning  the  milk  into  another 
flask.  It  is  rarely  necessary  to  adjust  the  reaction  of  milk  prepared  in 
this  way,  since,  if  acid  at  all,  it  is  usually  but  slightly  so.  If,  however, 
it  should  prove  more  than  1.5  per  cent  acid,  it  should  be  discarded 
or  neutrahzed  with  sodium  hydrate.  The  milk  may  then  be  poured 
into  test  tubes  without  further  additions,  or  litmus  solution  may  be 
added  in  a  quantity  sufficient  to  give  a  purplish  blue  color.  The 
tubes  are  sterilized  by  fractional  sterihzation  in  the  Arnold  sterihzer  for 
thirty  minutes  on  three  successive  days. 

Serum  Media. — Loeffler^s  Medium. — Beef  blood  is  collected  at  the 
slaughter  house  in  high  cylindrical  jars  holding  two  quarts  or  more. 
It  is  desirable  that  attempts  should  be  made  to  avoid  contamination 
as  much  as  is  feasible  by  previously  sterilizing  the  jars,  keeping  them 
covered,  and  exercising  care  in  the  collection  of  the  blood. 

The  blood  is  allowed  to  coagulate  in  the  jars,  and  should  not  be 
moved  from  the  slaughter  house  until  coagulated.  All  unnecessary 
shaking  of  jars  should  be  avoided.  As  soon  as  the  coagulum  is  fully 
formed,  adhesions  between  the  clot  and  the  sides  of  the  jar  should  be 
carefully  separated  with  a  sterile  glass  rod  or  wire.  The  jars  are  then 
set  away  in  the  ice  chest  for  24  to  36  hours.  At  the  end  of  this  time 
clear  serum  will  be  found  over  the  top  of  the  clot,  and  between  the  clot 
and  the  jar.  This  should  be  pipetted  off,  preferably  with  a  large  pipette 
of  50  to  100  c.c.  capacity,  or  siphoned  off  with  sterile  glass  tubing,  and 
transferred  to  sterile  flasks. 

To  three  parts  of  the  clear  serum  is  then  added  one  part  of  a  one 
per  cent  glucose  beef  infusion  or  veal  infusion  bouillon.  The  mixture  is 
filled  into  tubes,  perferably  the  short  test  tubes  commonly  used  for 
diagnostic  diphtheria  cultures.  The  tubes  are  then  placed  in  a  slanting 
position  in  the  apparatus  known  as  an  inspissator  (see  p.  71).  This  is 
a  double-walled  copper  box  covered  by  a  glass  lid,  cased  in  asbestos, 
and  surrounded  by  a  water  jacket.  It  is  heated  below  by  a  Bunsen 
flame.  Together  with  the  tubes  a  small  open  vessel  containing  water 
should  be  placed  in  the  inspissator  to  insure  sufficient  moisture.  The 
temperature  of  the  inspissator  is  now  raised  to  70°-75°  C,  care  being 
taken  that  the  rise  of  temperature  takes  place  slowly.  The  temperature 
is  maintained  at  this  point  for  two  hours,  and  the  process  is  repeated,  for 
the  same  length  of  time,  at  the  same  temperature,  on  six  successive 
days,  preferably  without  removing  the  tubes  from  the  inspissator  at 


132  BIOLOGY  AND  TECHNIQUE 

any  time.  It  is  also  possible,  though  less  regularly  yielding  good  results, 
to  sterilize  in  the  inspissator  for  one  day,  following  this  on  the  second 
and  third  days  by  exposure  for  thirty  minutes  to  100°  C.  in  the  Arnold 
steam  sterilizer.  In  doing  this,  the  Arnold  should  be  very  gradually 
heated,  at  first  without  outer  jacket,  this  being  lowered  only  after 
thorough  heating  has  taken  place. 

Serum-Water  Media  for  Fermentation  Tests. — For  the  deter- 
mination of  the  fermentative  powers  of  various  microorganisms 
for  purposes  of  differentiation,  Hiss  has  devised  the  following  media 
in  which  the  cleavage  of  any  given  carbohydrate  is  indicated, 
not  only  by  the  production  of  an  acid  reaction,  but  by  the  coagulation 
of  the  serum  proteids. 

Obtain  clear  beef  serum  by  pipetting  from  clotted  blood  in  the  same 
way  as  this  is  obtained  for  the  preparation  of  Loeffler's  blood-serum 
medium.  Add  to  this  two  or  three  times  its  bulk  of  distilled  water, 
making  a  mixture  of  serum  and  water  in  proportions  of  one  to  two  or 
three.  Heat  the  mixture  for  fifteen  minutes  in  an  Arnold  sterilizer  at 
100°  C.  to  destroy  any  diastatic  ferments  present  in  the  serum.  Add 
one  per  cent  of  a  five  per  cent  aqueous  litmus  solution  (the  varia- 
tion in  the  different  litmus  preparations  as  obtained  in  laboratories 
necessitates  a  careful  addition  of  an  aqueous  litmus  solution  until 
the  proper  color,  a  deep  transparent  blue,  is  obtained,  rather  than 
rigid  adherence  to  any  quantitative  directions).  Add  to  the  various 
fractions  of  the  medium  thus  made  one  per  cent  respectively  of  the 
sugars  which  are  to  be  used  for  the  tests. 

For  the  preparation  of  inulin  medium,  made  in  this  way  for  pneu- 
mococcus-streptococcus  differentiation,  it  is  necessary  to  sterilize  the 
inulin  dissolved  in  the  water  to  be  added  to  the  serum  in  an  autoclave 
at  high  temperature  (15  pounds  for  15  minutes)  in  order  to  kill  spores 
before  mixing  with  the  serum.  The  serum-water  media  are  sterilized  by 
the  fractional  method  at  100°  C,  at  which  temperature  they  remain  fluid. 

Special  Media  for  Colon-Typhoid  DifiEerentiation.^ — Hiss^  Plating 
Medium.^ — ^The  composition  of  this  medium  is  as  follows: 

Agar 15  gms. 

Gelatin 15     " 

Liebig's  meat  extract 5     " 

Sodium  chloride 5     " 

Dextrose 10     " 

Distilled  water 1,000  c.c. 

*  For  details  of  use  of  these  special  media  see  also  chapter  on  Bacillus  tj^hosus. 
»  HiaSf  Jour.  Exp.  Med.,  ii,  1897;  Jour.  Med.  Research,  viii,  1902. 


THE  PREPARATION  OF  CULTURE  MEDIA        133 

The  agar  is  thoroughly  dissolved  in  1,000  c.c.  of  distilled  water.  When 
the  agar  is  melted,  the  gelatin,  meat  extract,  and  salt  are  added  and  dis- 
solved by  further  heating.  Any  loss  in  weight  is  then  adjusted  by  the 
addition  of  water.  No  titration  or  adjustment  of  reaction  is  necessary. 
The  medium  should  be  cleared  with  the  whites  of  two  eggs,  and  filtered 
through  cotton.  To  the  cleared  medium  is  added  one  per  cent  of  dex- 
trose, and  the  medium  tubed,  about  8  c.c.  to  each  tube,  and  sterilized. 
Hiss'  Tube  Medium. — The  composition  is  as  follows: 

Agar 5  gms. 

Gelatin 80     " 

Liebig's  meat  extract 5    •" 

Sodium  chloride 5     " 

Dextrose 10     " 

Distilled  water 1,000  c.c. 

The  method  of  preparation  is  the  same  as  for  the  plating  medium.  The  agar 
is  thoroughly  dissolved,  and  then  the  gelatin,  meat  extract,  and  salt  are  added 
and  dissolved.  After  adjusting  the  loss  in  weight,  the  volume  should  be  deter- 
mined, a  careful  titration  made,  and  the  reaction  adjusted  to  one  and  five-tenths 
per  cent  acid  by  the  addition  of  ^  HCl  solution.  The  medium  is  cleared  with 
eggs,  filtered,  and  one  per  cent  dextrose  added.    It  is  then  tubed  and  sterilized. 

Hesse's  Medium} — The  medium  devised  by  Hesse  for  typhoid-colon 
differentiation  depends  for  its  usefulness,  as  does  the  Hiss  tube  medium, 
upon  the  great  motility  of  the  typhoid  bacillus.  It  may  be  used  directly 
for  the  examination  of  feces  or,  as  suggested  by  Jackson  and  Melia,^ 
after  preliminary  enrichment  of  the  material  to  be  examined  by  the  use 
of  the  lactose-bile  medium  of  Jackson.     (See  p.  138.) 

The  Hesse  medium  is  made  up  a^  follows: 

Agar 5  gms.  (4.5  gms.  absolutely  dry) 

Pepton  (Witte) 10     " 

Liebig's  beef  extract 5     " 

Sodium  chloride 8.5     " 

Distilled  water 1,000  c.c. 

Jackson  and  Melia  found  that  the  use  of  4.5  gms.  of  completely 
dried  agar  give  more  uniform  results,  as  the  amount  of  moisture  in 
commercial  agar  varies.     The  preparation  of  the  medium  is  as  follows: 

Dissolve  4.5  gms.  of  dry  agar  in  500  c.c.  of  distilled  water  over  a  free  flame, 
making  up  for  loss  by  evaporation.  In  another  vessel  10  gms.  of  pepton,  5  gms. 
of  beef  extract,  and  8.5  gms.  salt  are  dissolved  in  500  c.c.  distilled  water.  This 
may  be  heated  until  dissolved  and  loss  by  evaporation  made  up. 

*  Hesse,  Zeit.  f.  Hyg.,  Iviii,  1908.    ^  Jackson  and  Melia,  Jour,  of  Inf.  Dis.,  vi,  1909. 


1^4  .  BIOLOGY  AND  TECHNIQUE 

The  solutions  are  mixed  and  heated  thirty  minutes;  loss  by  evaporation  is 
then  made  up  and  the  solution  is  filtered  through  cotton.  The  reaction  is  ad- 
justed to  one  per  cent  acidity  and  the  medium  tubed — 10  c.c.  to  each  tube. 

The  typhoid  bacillus  is  characteristic  on  the  Hesse  medium  only  when  the 
dilution  poured  in  the  plates  is  so  high  that  only  a  few  colonies  appear.  The 
typhoid  colonies  are  much  larger  than  are  the  colon  colonies  and  may  often,  be 
several  centimeters  in  diameter. 

Conradi-Drigalshi  Medium} — Original  directions. 

(a)  Three  pounds  of  meat  are  infused  in  two  liters  of  water  for  twelve  hours 
or  more.  Strain,  boil  for  one  hour  and  add  20  gms.  Witte's  pepton,  20  gms.  of 
nutrose,  10  gms.  of  NaCl;  boil  one  hour  and  filter.  Add  60  gms.  of  agar.  Boil 
for  three  hours  (or  one  hour  in  an  autoclave)  until  agar  is  dissolved.  Render 
weakly  alkaline  to  litmus  paper,  filter,  and  boil  for  half  an  hour  more. 

(b)  Litmus  solution:  Two  hundred  and  sixty  c.c.  of  litmus  solution  are 
boiled  for  ten  minutes.  (The  htmus  solution  used  by  Conradi  and  Drigalski 
is  the  very  sensitive  aqueous  litmus  recommended  by  Kubel  and  Tieniann,  and 
purchasable  under  the  name.)  After  boiling,  30  grams  of  chemically  pure  lac- 
tose are  added  to  the  litmus  solution.  The  mixture  is  then  boiled  for  fifteen 
minutes,  and,  if  a  sediment  has  formed,  is  carefully  decanted. 

(c)  Add  the  hot  lactose  mixture  to  the  hot  agar  solution;  mix  well  and, 
if  necessary,  again  adjust  to  weak  alkaUne  reaction,  litmus  paper  used  as  an  in- 
dicator. To  this  mixture  add  4  c.c.  of  a  hot,  sterile  ten  per  cent  solution  of 
sodium  carbonate,  and  20  c.c.  of  a  freshly  made  solution  of  crystal  violet  (c. 
p.  Hochst),  0.1  gram  in  100  c.c.  of  sterile  distilled  water. 

Surface  smears  are  made  upon  large  plates.  These  are  incubated 
twenty-four  hours.  Typhoid  colonies  are  small,  blue,  and  transparent. 
Colon  colonies  are  large,  red,  and  opaque. 

Endows  Medium.'^ — 1.  Prepare  one  liter  of  meat  infusion  three  per  cent 
agar,  containing  10  grams  of  pepton  and  5  gr^ms  of  NaCl. 

2.  Neutralize  and  clear  by  filtration. 

3.  Add  10  c.c.  of  10%  sodium  carbonate  to  render  alkaline. 

4.  Add  10  grams  of  chemically  pure  lactose. 

5.  Add  5  c.c.  of  alcoholic  fuchsin  solution,  filtered  before  using.  (Endo  in 
his  original  contribution  does  not  mention  the  strength  of  this  fuchsin  solu- 
tion, which,  however,  should  be  saturated.    This  colors  the  medium  red. 

6.  Add  25  c.c.  of  a  10%  sodium  sulphite  solution.  This  again  decolorizes 
the  medium,  the  color  not  entirely  disappearing,  however,  until  the  agar  is 
cooled. 

7.  Put  into  test  tubes,  15  c.c.  each,  and  sterilize. 

1  Conradi-Drigalski,  Zeit.  f.  Hyg.,  xxxix,  1902. 

2  Endo,  Cent.  f.  Bakt.,  xxxv,  1904. 


THE  PREPARATION  OF  CULTURE  MEDIA  135 

The  medium  should  be  kept  in  dark.  Plates  are  poured  and  surface  smears 
made.    The  typhoid  colonies  remain  colorless,  while  those  of  coli  become  red. 

The  preparation  of  Endo's  medium  presents  difficulties  due  to  the 
varying  purity  of  sodium  sulphite.  Kastle  and  Elvove  ^  recommend  the 
use  of  anhydrous  sodium  sulphite  instead  of  the  crystallized  variety. 
Harding  and  Ostenberg  ^  add  sodium  sulphite  solution  to  a  measured 
amount  of  .5  per  cent  f  uchsin  to  determine  the  proportions  which  give  the 
greatest  delicacy  of  reaction  as  tested  with  formaldehyde.  The  propor- 
tions so  determined  are  then  added  to  the  hot  3  per  cent  agar. 

Although  Endo  described  his  medium  as  dependent  upon  the  forma- 
tion of  acid  by  the  bacteria,  this  is  not  so.  Acids  give  no  coloration  of 
the  sulphite-fuchsin  mixture.  Indeed  this  mixture  is  used  by  chemists 
under  the  name  of  Schiff 's  reagent  as  a  test  for  aldehydes.  Acids  decol- 
orize the  red  caused  by  aldehydes,  and  this  accounts  for  the  frequent 
late  discoloration  of  red  colon  colonies  on  prolonged  cultivation.  The 
medium  is  red  when  hot,  and  colorless  when  cold,  because  the  com- 
pound between  sulphite  and  f uchsin  dissociates  in  the  hot  solution. 

Kendall's  Modification  of  Endows  Medium.^ — 1.5  per  cent  meat  extract  agar 
is  prepared,  and  the  reaction  adjusted  faintly  alkaline  to  litmus  by  the  addition 
of  NaOH.  This  agar  is  stored  in  small  flasks  and  it  is  usually  convenient  to 
keep  flasks  containing  100  c.c.  each.  Just  before  use,  1  per  cent  of  lactose  is 
added,  and  then  decolorized  fuchsin  solution,  as  in  Endows  medium.  Add 
about  1  c.c.  of  decolorized  fuchsin  solution,  made  up  as  above  by  mixing  roughly 
prepared  10  per  cent  sodium  sulphite  with  saturated  alcoholic  fuchsin.  (The 
proportions  of  fuchsin  and  sulphite  are  sometimes  difficult  to  adjust,  possibly 
by  reason  of  impurities  in  the  sulphite  due  to  formation  of  sulphate.  The  in- 
structions given  by  most  workers  at  present  are  to  use  10  c.c.  of  a  10  per  cent 
aqueous  solution  of  sodium  sulphite,  a§d  to  add  to  this  1  c.c.  of  a  10  per  cent 
solution  of  fuchsin  in  96  per  cenf  alcohol.)  When  these  flasks  containing  the 
various  ingredients  are  hot  they  are  red  or  pink,  but  when  plates  are  poured 
and  allowed  to  harden,  the  medium  should  be  either  colorless  or  very  faintly 
pinkish.  It  is  best  to  pour  a  number  of  plates  rather  thickly  and  then  allow 
them  to  dry  with  the  covers  off.  Inoculations  from  the  feces  solution  are  then 
made  by  surface  smear,  with  a  bent  glass  rod.  Colon  colonies  are  pinkish  and 
red;  typhoid  colonies,  smaller  and  grayish. 

In  concluding  the  description  of  some  of  the  most  important  typhoid  isola- 
tion media,  we  would  like  to  add  that  a  great  deal  seems  to  depend  upon  the 

^  Kastle  and  Elvove,  Jour.  Inf.  Dis.,  xvi,  1909. 

2  Harding  and  Ostenberg,  Jour,  of  Inf.  Dis.,  xi,  1,  1909, 

3  Kendall,  Boston  Med.  &  Surg.  Jour. 


136  BIOLOGY  AND  TECHNIQUE 

habit-acquired  skill  which  the  individual  worker  attains.  None  of  these  stool 
isolation  media  are  ordinarily  successful  at  once  in  the  hands  of  anyone,  and  a 
certain  amount  of  practice  must  be  attained  before  one  can  judge  of  the  use- 
fulness or  uselessness  of  a  medium. 

Brilliant  Green  Agar  for  Typhoid  Isolation. — Krumwiede  has  recently 
devised  a  brilliant  green  agar  with  which  he  has  had  excellent  results.* 
The  basis  is  an  extract  agar  like  that  used  for  Endo's  medium: 

Beef  Extract 0.3% 

Salt 0.5% 

Peptone 1.0% 

Agar 1.5% 

(Domestic  peptones  are  satisfactory.) 

Dissolve  in  autoclave;  clear  and  filter.  A  clear  agar  is  essential.  The  final 
reaction  of  the  medium  is  to  be  neutral  to  ^  Andrade's  indicator,  which  in  terms 
of  phenolphthalein  is  0.6-0.7%  acid  (normal  HCl).  It  is  more  convenient  to 
,have  the  reaction  set  slightly  alkaline  to  litmus  at  the  time  of  preparation  and 
to  acidify  each  bottle  as  used.  The  agar  is  bottled  in  100  c.c.  amounts  and  auto- 
claved.  When  needed,  the  bottles  are  melted  and  the  volume  of  each  cor- 
rected (if  necessary)  to  an  approximate  100  c.c.    Add  to  each  bottle: 

One  per  cent  Andrade's  Indicator. 

Acid  to  bring  to  neutral  point  of  the  indicator.' 

One  per  cent  Lactose.* 

0.1  per  cent  Glucose.* 

Brilliant  Green  in  0.1  per  cent  aqueous  solution. 

Two  dilutions  of  dye  are  used  in  routine  plating,  corresponding  to  1-500,000 
and  1-330,000  in  terms  of  soHd  dye  (0.2  c.c.  and  0.3  c.c.  of  0.1  per  cent  solution 
per  100  c.c.  of  agar).  The  sample  of  dye  which  Krumwiede  has  used  is  from 
Bayer,  but  he  has  also  tested  and  found  equally  satisfactory  samples  from 
Griibler  and  Hochst.  0.1  gram  of  dye  S  accurately  weighed  on  a  foil,  washed 
with  boiling  H2O  into  a  100  c.c.  volumetric  Iflask  and  made  up  to  the  mark 
when  cool.  The  flask  should  be  clean  and  neutral  (by  test).  Fresh  solutions 
vary  in  activity  (see  standardization  tests);  they  keep  about  a  month. 

1  We  are  indebted  to  Dr.  Krumwiede  for  a  preliminary  account  of  this  method. 

^  Andrade's  Indicator:  0.5  per  cent  aqueous  acid  fuchsin 100  c.c. 

Normal  NaOH 16  c.c. 

The  dye  is  slowly  (2  hours)  alkalinized  to  the  color-base;  the  red  tint  is  restored  by 
acids. 

3  An  agar  is  neutral  to  Andrade  when,  hot,  the  color  is  a  deep  red,  but  fades  com- 
pletely on  cooling.  This  is  determined  by  cooling  3  or  4  c.c.  of  acidified  hot  agar 
in  a  serum  tube  under  the  tap  and  adjusting  accordingly. 

*  These  are  conveniently  added  from  one  sterile  solution  containing  20%  lactose 
and  2%  dextrose,  5  c.c.  to  100  of  agar  gives  the  requisite  concentration. 


THE  PREPARATION  OF  CULTURE  MEDIA  137 

Each  bottle  is  mixed  and  poured  into  six  plates  only  (a  thick  layer  of  agar 
gives  the  most  characteristic  colonies).  Plates  are  left  micovered  until  agar 
has  "jellied";  porous  tops  are  used;  dry  plates  are  essential  to  avoid  diffusion. 

Standardization:  The  agar  must  have  proper  "balance."  The  reaction  is 
important;  sediment  reduces  the  activity  of  the  dye  and  light  colored  media 
are  better  than  darker  ones.  Different  lots  of  agar  with  the  same  dye  solution 
act  ununiformly;  a  new  batch  or  a  new  solution  must  be  tested. 

Any  variation  in  the  composition  of  the  media  necessitates  a  readjustment 
of  dye  concentration;  this  statement  cannot  be  over-emphasized. 

Brilliant  green,  in  appropriate  dilutions,  not  only  inhibits  all  Gram- 
positive  and  many  Gram-negative  bacteria,  but  exhibits  differential 
action  on  the  colon-typhoid  group.  Paratyphoid  and  the  B.  lactis 
aerogenes  are  untouched,  typhoid  is  restrained  only  at  low  dilutions, 
while  dysentery  and  the  other  colon  group  are  extremely  susceptible. 
The  typhoid  colony  on  this  medium  is  characteristic.  Looking  through 
the  plate  against  a  dark  surface,  in  oblique  light  the  colony  has  a  snow- 
flake  appearance;  the  edge  delicately  serrate.  With  artificial  light  and 
a  hand  lens,  the  texture  is  that  of  a  coarse  woolen  fabHc.  Acid  produc- 
tion from  the  trace  of  glucose  may  tinge  the  colony.    The  colony  is  large. 

Malachite-Green  Bouillon  (Peabody  and  Pratt).' — To  100  c.c.  of  beef  in- 
fusion broth  add  10  c.c.  of  one  per  cent  solution  of  malachite  green,  Hochst 
120,  made  with  sterile  water.    This  is  tubed. 

This  medium  is  used  as  an  enriching  fluid.  One  drop  of  the  suspected 
material  (emulsified  stool)  is  added  to  each  tube  and  after  incubation 
for  eighteen  to  twenty-four  hours  inoculations  may  be  made  upon  plates. 

Peabody  and  Pratt  found  a  reaction  of  .5  per  cent  acidity  to  phenol- 
phthalein  most  favorable. 

Bile  Medium^. — ^ (Recommended  -for  blood  cultures  by  Buxton  and 
Coleman.)     The  medium  is  prepared  as  follows: 

Ox-bile 900  c.c. 

Glycerin 100  c.c. 

Pepton 20  grams 

Put  into  small  flasks  containing  quantities  of  about  100  c.c.  each  and  sterilized 
by  fractional  sterilization. 

Jackson's  Lactose-Bile  Medium.^ — This  medium  is  of  great  use  in 
isolating  B.  typhosus  and  B.  coli  from  water,  and  serves  as  a  valuable 
enriching  medium  in  isolating  them  from  other  sources.     Jackson  and 

^  Peabody  and  Pratt,  Boston  Med.  and  Surg.  Jour.,  clviii,  7,  1908. 

2  Canradi,  Deut.  med.  Woch.,  32,  1906. 

«  Jackson,  "Biol.  Studies  of  Pupils  of  W.  T.  Sedgwick,"  1906,  Univ.  Chicago  Press. 


138  BIOLOGY  AND  TECHNIQUE- 

Melia  ^  found  that  in  this  medium  B.  typhosus  and  B.  coli  outgrow  all 
other  microorganisms  and  eventually  B.  typhosus  will  even  outgrow  B.  coli. 
It  consists  of  sterilized  undiluted  ox-bile  (or  a  ten  per  cent  solution  of  dry, 
fresh  ox-bile)  to  which  is  added  one  per  cent  pepton  and  one  per  cent  lactose. 
It  is  filled  into  fermentation  tubes  and  sterihzed  by  the  fractional  method. 

MacConkey's  Bile-Salt  Agar. — 

Sodium  glycocholate 0.5  per  cent. 

Pepton 2.0    "      " 

Lactose 1.0    "      " 

Agar 1.5"      " 

Tap  water q.s. 

The  agar  and  pepton  are  dissolved  and  cleared  and  the  lactose  and 
sodium  glycocholate  added  before  tubing.  The  B.  typhosus  produces  no 
change;  B.  coli,  producing  acid,  causes  precipitation  of  the  bile  salts. 

Neutral-Red  Medium. — To  100  c.c.  of  a  one  or  two  per  cent  glucose  agar  add 
1  c.c.  of  a  saturated  aqueous  solution  of  a  neutral-red. 

The  medium  is.  used  in  tubes,  stab  or  shake  cultures.  The  typhoid  bacillus 
produces  no  change,  while  members  of  the  colon  group  render  the  medium  color- 
less by  reduction  of  the  neutral-red  and  produce  gas. 

Barsiekow's  Medium.^ — To  200  c.c.  of  cold  water,  add  10  grams  of  nutrose 
and  allow  to  soak  for  one-half  to  one  hour.  Pour  this  into  800  c.c.  of  boiling 
water,  and  heat  for  twenty  minutes  in  an  Arnold  sterihzer  at  100°  C.  Filter 
through  cotton  and  to  the  opalescent  solution  of  nutrose  add  5  grams  of  NaCl, 
10  grams  of  lactose,  and  sufficient  aqueous  litmus  solution  to  give  a  pale  blue 
color.  3 

RusselVs  Double  Su^ar  Agar.* — Russell  has  devised  a  simple  medium  for 
quick  identification  of  typhoid  bacilli. 

A  2%  or  3%  extract  agar  is  used,  about  0.8%  acid  to  phenolphthalein  0.8%. 
Enough  litmus  solution  is  added  to  give  it  the  ordinary  deep  blue.  The  re- 
action is  then  adjusted  with  sodium  hydrate  until  neutral  to  litmus.  Finally 
1%  lactose  and  0.1%  glucose  (dissolved  in  a  small  amount  of  hot  water)  are 
added,  the  medium  is  carefully  sterilized  in  the  Arnold  sterilizer  and  slanted. 
Inoculations  are  made  by  surface  streak  and  stab.  The  typhoid  bacillus  grows 
colorless  on  the  surface,  but  under  the  imperfect  anaerobic  conditions  at  the 
bottom  of  the  stab,  a  bright  red  color  is  developed  by  acid  formation. 

Dieudonne^s  Selective  Medium  for  cholera  spirillum.    See  page  584. 
Enriching  Substances  Used  in  Media. — For  the  cultivation  of  micro- 

1  Jackson  and  Melia,  Jour.  Inf.  Dis.,  vi,  1909. 

2  Barsiekow,  Wien.  klin.  Rund.,  xliv,  1901. 

3  Filtration  may  be  done  through  paper,  but  takes  a  long  time. 
*  Russell,  Jour.  Med.  Research,  xxv,  1911,  217. 


THE  PREPARATION  OF  CULTURE  MEDIA  139 

organisms  which  are  sensitive  to  their  food  environment,  it  is  often  neces- 
sary or  advisable  to  add  to  the  ordinary  media  enriching  substances, 
which  empirical  study  has  shown  to  favor  the  growth  of  the  organism 
in  question.  The  substances  most  commonly  used  for  such  enrichment 
are  glucose,  nutrose  (sodium  caseinate),  glycerin,  sodium  formate,  and 
unsohdified  animal  proteids.  As  animal  and  blood  serum  and  whole 
blood  must  frequently  be  used  in  this  way,  an  understanding  of  the 
methods  employed  in  obtaining  these  substances  is  necessary. 

Method  of  Obtaining  Blood  and  Blood  Media. — Blood  serum  from 
beef  and  sheep  may  be  collected  in  the  manner  recommended  for  the 
collection  of  such  serum  in  the  preparation  of  Loeffler's  medium,  pipetted 
into  test  tubes,  and  sterilized  in  the  fluid  state  by  exposure  to  tempera- 
tures ranging  from  60°  to  65°  C,  for  one  hour  upon  six  consecutive  days. 
It  is  not  a  simple  matter  to  sterilize  serum  in  this  way  and  requires  much 
time  and  care. 

The  method  most  commonly  employed,  in  laboratories  which  have 
access  to  hospitals,  for  obtaining  clear  serum  depends  upon  the  collection 
of  exudate  or  transudate  fluids  by  sterile  methods  directly  from  the 
pleural  cavity,  the  abdominal  cavity,  or  the  hydrocele  cavity.  Sterile 
flasks  or  test  tubes  are  prepared  and  the  fluid  is  allowed  to  flow  directly 
out  of  the  cannula  into  these.  It  is  necessary  to  avoid  carbolic  acid  or 
other  disinfectants  in  sterilizing  instruments  and  rubber  tubing  used 
during  the  operation.  These  should  be  brought  into  the  ward  in  the 
water  in  which  they  have  been  boiled  and  not  in  strong  antiseptic  solu- 
tions, as  is  frequently  done.  The  fluid  so  obtained  may  be  incubated 
and  the  contaminated  tubes  discarded.  The  serum  may  then  be  added, 
in  proportions  of  one  to  three,  to  sterile  broth  or  melted  agar. 

Agar  thus  used  is  melted  and  cooled  to  60°  C,  or  below.  One-third 
of  its  volume  of  warmed  exudate  fluid  is  added,  and  the  plates  are 
poured. 

Serum  may  be  rendered  free  of  bacteria  by  filtration  through  a 
Berkefeld  or  Pasteur-Chamberland  filter.  This  is  an  effectual  method, 
but  requires  much  time  and  care. 

Whole  blood  may  be  obtained  for  cultural  purposes  by  bleeding 
rabbits  or  dogs  or  other  animals  directly  from  a  blood-vessel  into  tubes  of 
melted  agar.  In  the  case  of  a  rabbit,  after  the  administration  of  an  anes- 
thetic (ether),  an  incision  is  made  directly  over  thie  trachea,  and,  by 
careful  section,  the  carotid  artery  is  isolated,  lying  close  to  the  side  of 
the  trachea. 


140  BIOLOGY  AND  TECHNIQUE 


THE  INFLUENCE  OP  DYE  STUFFS  UPON  BACTERIAL  GROWTH, 
AND  AS  ADDITIONS  TO  SELECTIVE  MEDIA 

In  describing  the  selective  media  for  typhoid  bacilU  we  have  seen 
that  malachite  green  and  crystal  violet  have  been  found  to  exert  a 
certain  amount  of  selective  action  upon  the  tjrphoid  and  colon  groups. 
The  selective  influence  of  various  dyes  has  been  recently  again  studied 
by  Churchman.  Churchman  ^  found  that  the  addition  of  gentian 
violet  in  dilutions  of  1 :  100,000,  to  media,  inhibited  some  bacteria,  while 
others  grew  luxuriantly  in  its  presence.  Extremely  interesting,  both 
practically  and  theoretically,  is  his  observation  that  upon  such  gentian 
violet  media  bacteria  fall  into  two  groups.  Those  which  grow  on 
gentian  violet  correspond  in  a  general  way  to  the  Gram-negative  bacteria; 
those  which  fail  to  develop  on  these  media  correspond  roughly  with  the 
Gram-positive  species.  One  strain  of  the  enteritidis  group  could  not 
be  cultivated  on  gentian  violet,  and  this  was  found  to  differ  from  the 
others  also  in  its  agglutination  tests. 

Signorelli  ^  claims  that  dahUa  is  useful  in  differentiating  true  cholera 
strains  from  similar  spirilla.  The  true  cholera  strains  grew  with  colored 
colonies,  while  others  remain  colorless,  in  his  experiments. 

Krumwiede  and  Pratt  ^  were  unable  recently  to  confirm  the  claims 
of  Signorelli.  However  they  fully  confirm  the  findings  of  Churchman 
both  as  to  the  selective  action  of  gentian  violet  and  in  regard  to  the 
classification  of  bacteria  into  two  groups  corresponding  to  their  reaction 
to  the  Gram  stain.  They  state  that  among  Gram-negative  bacteria  a 
strain  is  occasionally  found  which  will  not  grow  on  the  gentian  violet 
media,  differing  in  this  respect  from  other  members  of  the  same  species. 
They  find  also  that  the  reaction  is  quantitative. 

The  streptococcus-pneumococcus  group,  according  to  their  investi- 
gations, differs  from  other  bacteria  in  being  able  to  grow  in  the  presence 
of  quantities  of  violet  which  inhibit  other  Gram-positive  species.  Dys- 
entery bacilli  show  variations.  Other  dyes  which  they  investigated 
showed  no  specific  inhibitory  properties  which  could  be  utilized  for 
classification. 

1  Churchman,  Jour.  Exp.  Med.,  16,  1912;  also  Churchman  and  Michael,  ibid. 

2  Sigruyrelli,  Centralbl.  f .  Bakt.,  Orig.  56,  1912. 

^Krumwiede  and  Pratt,  Centralbl.  f.  Bakt.,  Orig.  68,  1913;  and  Proc.  N.  Y. 
Path.  Soc,  xiii,  1913. 


CHAPTER  VIII 
METHODS  USED  IN  THE  CULTIVATION  OF  BACTERIA 

INOCULATION   OF    MEDIA 

The  transference  of  bacteria  from  pathological  material  to  media, 
or  from  medium  to  medium,  for  purposes  of  cultivation,  is  usually  ac- 
complished by  means  of  a  platinum  wire  or  loop.  The  platinum  wire 
used  should  be  thin  and  yet  possess  a  certain  amount  of  stiffness  and  be 
about  two  to  three  inches  in  length.  This  is  fused  into  the  end  of  a  glass 
rod  six  to  eight  inches  long.  It  is  an  advantage,  though  not  necessary, 
to  use  rods  of  so-called  "seahng-in"  glass  which,  having  the  same  co- 
efficient of  expansion  as  platinum,  does  not  crack  during  sterilization. 
For  work  with  fluid  media,  the  wire  should  be  bent  at  its  free  end 
so  as  to  form  a  small  loop  which  will  pick  up  a  drop  of  the  liquid.  For 
the  inoculation  of  solid  media  and  the  making  of  stab  cultures,  a  straight 
"needle "  or  wire  should  be  used.  Other  shapes  of  these  wires  and  spat- 
ulae  from  heavy  wire  have  been  devised  for  various  purposes  and  are 
easily  improvised  as  occasion  demands.      (See  Fig.  27.) 

When  making  a  transfer  from  one  test  tube  to  another,  the  tubes 
should  be  held  between  the  thumb  and  first  and  second  fingers  of  the 
left  hand,  as  shown  in  Fig.  28.  The  plugs  are  then  removed  by  grasping 
them  between  the  small  and  ring  fingers  and  ring  and  middle  fingers  of 
the  right  hand,  first  loosening  any  possible  adhesions  between  glass  and 
plugs  by  a  slight  twisting  motion.  The  platinum  wire  is  held  meanwhile 
by  the  thumb  and  index  fingers  of  the  right  hand  in  the  manner  of  a  pen. 
The  wire  is  heated  red  hot  in  a  Bunsen  flame,  and  is  then  passed  into  the 
culture  tube  without  being  allowed  to  touch  the  glass.  It  is  held  sus- 
pended within  the  tube  for  a  few  seconds  to  permit  of  cooling  before 
touching  the  bacterial  growth.  The  wire  is  then  allowed  to  touch  lightly 
the  surface  of  the  growth  and  a  small  amount  is  picked  up.  (See  Fig. 
29.)  It  is  then  removed  from  the  tube  without  allowing  it  to  touch  the 
sides  of  the  glass,  and  is  passed  into  the  tube  which  is  to  be  inoculated. 
If  the  tube  contains  a  slanted  medium,  such  as  agar,  a  light  stroking 
motion  from  the  bottom  of  the  slant  to  its  apex  will  deposit  the  bacteria 

141 


142 


BIOLOGY  AND  TECHNIQUE 


/T^ 


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rl 


13 


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fl 


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upon  the  medium  evenly  along  a  central  line.  The  needle  may  also  b^ 
plunged  downward  into  the  substance  of  the  nutritive  material  so 
that  in  the  same  tube  both  surface  growth  and  deep  growth  may  be 
observed.    If  a  stab  culture  is  to  be  made  in  unslanted  agar  or  in  gelatin, 

the  needle  is  simply  plunged  straight 
downward  as  nearly  as  possible  along 
the  axis  of  the  medium.  If  a  fluid 
medium  is  being  inoculated,  the  wire 
should  be  introduced  only  into  the 
upper  part  of  the  liquid  and  the  bac- 
teria gently  rubbed  into  emulsion 
against  the  side  of  the  glass.  The 
needle  is  then  removed  from  the  tube, 
the  stopper  carefully  replaced,  and  the 
platinum  wire  immediately  sterilized  in 
the  flame.  This  sterilization  of  plati- 
num needles  after  they  have  been  in 
contact  with  bacteria  should  become 
second  nature  to  those  working  with 
bacteria,  since  an  infraction  against 
this  rule  may  give  rise  to  serious  and 
widespread  consequences.  In  burning 
off  platinum  needles  it  is  well  to  re- 
member that  a  part  of  the  glass  rod, 
as  well  as  the  wire  itself,  is  introduced 
into  the  tubes  and  may  become  con- 
taminated, and  for  this  reason  the 
rod  itself,  at  least  in  its  lower  two  or 
three  inches,  should  be  passed  through 
the  flame  as  well  as  the  wire.  As  an 
extra  precaution  against  contamina- 
tion, the  lips  of  test  tubes  and  flasks  and  the  protruding  edges  of  cotton 
plugs  may  be  passed  through  the  flame  and  singed. 

THE   ISOLATION   OF   BACTERIA   IN    PURE    CULTURE 


Fig.  27. — Platinum  Wires. 


It  is  obvious  that  in  many  cases  where  bacteria  are  cultivated  from 
water,  milk,  pathological  material,  or  other  sources,  many  species  may 
be  present  in  the  same  specimen.  It  is  likewise  obvious  that  scientific 
bacteriological  study  of  any  bacterium  can  be  made  only  if  we  obtain 


METHODS   USED   IN   CULTIVATION   OF   BACTERIA 


143 


this  particular  species  entirely  apart  from  others,  in  what  is  known  as 
"pure  culture."  The  earliest  methods  for  accomplishing  this  were  the 
methods  of  Pasteur  and  of  Cohn  who  depended  upon  the  power  of  one 
species  to  outgrow  all  others,  if  cultivated  for  a  sufficient  length  of  time 
in  fluid  media.  This  method,  of  course,  was  inadequate  in  that  it  was 
often  purely  a  matter  of  chance  which  one  of  the  mixture  of  species 
was  finally  obtained  by  itself.  A  later  method,  by  Klebs,  depends 
upon  serial  dilution,  in  test  tubes  of  fluid  media,  by  which  the  eventual 
transference  of  one  germ  only,  to  the  last  tube  was  attempted.  Such 
methods,  none  of  them  of  great  practical  value,  have  been  entirely  dis- 


FiG.  28. — Taking  Plugs  from  Tubes  before  Inoculation. 


placed  by  those  made  possible  by  Koch's  introduction  of  solid  media 
which  may  be  rendered  fluid  by  heat. 

The  methods  now  employed  for  the  isolation  of  bacteria  depend  upon 
the  inoculation  of  gelatin  or  agar,  when  in  the  melted  state,  the  thorough 
distribution  of  the  bacteria  in  these  liquids  by  mixing,  and  the  sub- 
sequent congealing  of  these  media  in  thin  layers.  By  this  means  the  in- 
dividual bacteria,  distributed  in  the  medium  when  liquid,  are  held  apart 
and  separate  when  the  medium  becomes  stiff.  The  masses  of  growth 
or  "colonies"  which  develop  from  these  single  isolated  microorganisms 
are  discrete  and  are  descendants  of  a  single  organism,  and  can  be  trans- 
ferred, by  means  of  a  process  known  as  "  colony-fishing,"  to  fresh  sterile 

culture  media. 
11 


144  BIOLOGY  AND  TECHNIQUE 

Plaitng.— The  first  method  employed  by  Koch  for  bacterial 
isolations  was  one  that  consisted  in  the  use  of  srniple  plates  of  glass, 
about  3x4  inches  in  size,  mounted  upon  a  leveling  stand.  A  wooden 
triangle,  supported  upon  three  adjustable  screw-feet,  formed  the  base  of 
this  apparatus.  Upon  this  was  set  a  covered  crystallizing  dish  which 
could  be  filled  with  ice  water.  Upon  the  top  of  this  rested  the  sterilized 
plates  under  a  bell  j  ar.  By  screwing  up  or  down  upon  the  supports  of  the 
triangle,  leveling  of  the  plate  could  be  achieved  and  controlled  by  a  spirit- 
level  placed  at  its  side.    The  inoculated  gelatin  was  poured  upon  the 


Fig.  29. — Inoculating. 

plate  and  spread  and  rapidly  cooled  and  hardened  by  the  cold  water 
contained  in  the  crystallizing  dish. 

The  original  method  of  Koch  has  been  modified  considerably  and  the 
method  universally  employed  at  present  depends  upon  the  use  of  circular 
covered  dishes,  the  so-called /*ein  dishes.  These  obviate  the  necessity  of 
a  leveling  stand  and  prevent  contamination  of  the  plate  when  once 
poured.  Each  Petri  dish  plate  consists  of  two  circular  glass  dishes;  the 
smaller  and  bottom  dish  has  an  area  of  63.6  square  centimeters;  the 
larger  is  used  as  a  cover  for  the  smaller,  and  forms  a  loosely  fitting  lid. 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA  145 

The  plates  when  fitted  together  are  sterilized  and  thus  form  a  closed 
cell  which,  if  properly  handled,  may  remain  sterile  indefinitely. 

The  technique  for  making  a  pour  plate  for  the  purpose  of  isolating 
bacteria  from  mixed  culture  is  as  follows: 

The  actual  "pouring"  of  plates  is  preceded  by  the  preparation  of 
usually  three  graded  dilutions  of  the  material  to  be  examined.  For  this 
purpose  three  agar  or  gelatin  tubes  are  melted  and,  in  the  case  of  the  agar, 
are  cooled  to  a  temperature  of  about  42°  C.  in  a  water  bath.  A  platinum 
loopf ul  of  the  material  to  be  examined  is  transferred  to  one  of  these  tubes. 
The  bacteria  are  then  thoroughly  distributed  throughout  the  melted 


Fig.  30. — Pouring  Inoculated  Medium  into  Petri  Plate. 

gelatin  or  agar  by  alternately  depressing  and  raising  the  plugged  end 
of  the  tube,  giving  it  a  rotary'  motion  at  the  same  time.  This  thoroughly 
distributes  the  bacteria  throughout  the  medium  without  allowing  the 
formation  of  air-bubbles.  Two  loopfuls  of  this  mixture  are  then  trans- 
ferred to  the  second  tube  and  a  similar  mixing  process  is  repeated. 
This  second  tube  contains  the  bacteria  in  much  greater  dilution  than 
the  first  and  the  colonies  which  will  form  in  the  plate  poured  from  this 
tube  will  be  farther  apart.  A  third  dilution  is  then  made  by  transferring 
five  loopfuls  of  the  mixture  in  the  second  tube  to  the  third.  This  again 
is  mixed  as  before.    The  contents  of  the  tubes  are  then  poured  into  three 


146  BIOLOGY  AND  TECHNIQUE 

sterile  Petri  dishes.  The  pouring  should  be  done  with  great  care. 
The  cover  of  the  dish  is  raised  along  one  margin  simply  far  enough  to 
permit  the  insertion  of  the  end  of  the  test  tube,  the  plug  of  which  has 
been  removed  and  the  lips  passed,  with  a  rotary  movement,  through  the 
flame.  The  medium  is  poured  into  the  dish  without  the  lips  of  the 
tube  being  allowed  to  touch  either  the  bottom  or  the  cover  of  the  dish. 
The  cover  is  then  replaced  and  the  medium  allowed  to  harden. 

When  agar  has  been  used,  the  dishes  may  be  placed  in  an  incubator 
at  37°  C.  It  is  well  to  place  the  plates  upside  down  in  the  incubator. 
This  prevents  the  condensation  water,  squeezed  out  of  the  agar  dur- 
ing hardening,  from  collecting  on  its  surface,  and  forming  channels  for 
the  diffuse  spreading  of  bacteria.  The  same  end  may  be  attained  by  the 
use  of  Petri  plates  provided  with  porous  earthenware  lids,  as  suggested 
by  Hill.  Simple  inversion  of  the  plates,  however,  usually  suffices.  When 
gelatin  has  been  used,  the  plates  are  allowed  to  remain  in  a  dark  place  at 
room  temperature  or  in  a  special  thermostat  kept  at  22°-25°  C. 

Colonies  in  agar,  kept  at  37.5°  C. ,  usually  develop  in  eighteen  to  twenty- 
four  hours;  those  in  gelatin  or  agar  at  room  temperature  in  from  twenty- 
four  to  forty-eight  hours,  depending  upon  the  species  of  bacteria  which 
are  being  studied.  Often  in  the  second  dilution,  more  frequently  in 
the  third,  the  colonies  will  be  found  well  apart  and  can  then  be  "fished." 
The  process  of  "  colony-fishing  "  is  one  which  requires  practice  and  should 
always  be  done  with  care,  for  upon  its  success  depends  the  purity  of  the 
sub-culture  obtained.  Colonies  should  never  be  fished  under  the  naked 
eye,  no  matter  how  far  apart  and  discrete  they  may  appear,  since  not 
infrequently  close  to  the  edge  of  or  just  beneath  a  larger  colony  there 
may  be  a  minute  colony  of  another  species  which  may  be  too  small  to  be 
visible  to  the  naked  eye,  but  which,  nevertheless,  if  touched  by  accident 
will  contaminate  the  sub-culture. 

For  proper  "  fishing,'^  the  Petri  plate  with  cover"removed,  should  be 
placed  upon  the  stage  of  the  microscope  and  examined  with  a  low  power 
objective,  such  as  Leitz  No.  2  or  Zeiss  AA.  The  steriHzed  platinum 
needle,  held  in  the  right  hand,  is  then  carefully  directed  into  the  line 
of  focus  of  the  lens,  while  the  small  finger  of  the  hand  is  steadied  upon 
the  edge  of  the  microscope  stage.  When  the  point  of  the  needle  is 
clearly  visible  through  the  microscope,  it  is  gently  depressed  until  it 
is  seen  to  touch  the  colony  and  to  carry  away  a  portion  of  it.  The 
needle  is  then  withdrawn  without  again  touching  the  nutrient  medium 
or  the  edges  of  the  glass  or  the  lens,  and  transferred  to  a  tube  of  what- 
ever medium  is  desired.    In  this  way,  individuals  of  one  colony,  de- 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA 


147 


scendants  of  a  single  bacterium  of  the  original  mixture, — are  carried 
over  to  the  fresh  medium. 

Esmarch  Roll  Tubes. ^ — A  simple  method  of  obtaining  separate  colo- 
nies is  that  devised  by  von  Esmarch  and  known  as  "  roll-tube  "  cultiva- 
tion. Tubes  of  melted  gelatin  are  inoculated  with  various  dilutions  of 
the  bacterial  mixture  and,  while  still  liquid,  are  laid  in  an  almost  horizon- 


FiG.  31. — Streak  Plate. 

tal  position  upon  a  block  of  ice,  which  has  been  grooved  slightly  by 
means  of  a  test  tube  filled  with  hot  water.  The  test  tube  containing  the 
gelatin,  after  being  placeji  in  this  groove,  is  rapidly  revolved  by  passing 
the  fingers  of  the  right  hand  across  it,  while  its  base  is  carefully 
steadied   with   the   left   hand.     If  the    revolving    is   carried   out   with 


Esmarch,  Zeit.  f.  Hyg.,  i,  1886. 


148 


BIOLOGY  AND  TECHNIQUE 


sufficient  speed,  the  gelatin  will  harden  in  a  thin  layer  on  the  inner 
surface  of  the  tube.  The  colonies  will  develop  in  this  layer  and  may  be 
"  fished  "  by  means  of  a  platinum  wire  with  bent  point  introduced  into  the' 
tube.  This  method  is  useful  for  certain  purposes,  but  is  too  inconvenient 
for  routine  work.    It  is  now  rarely  used. 

Separatio:  i  of  Bacteria  by  Surface  Streaking. — When  it  is  necessary  to 
isolate  bacteria  like  the  gonococcus.  Bacillus  influ- 
enzae, the  pneumococcus,  and  others,  which,  because 
of  great  sensitiveness  to  environment  and  possibly  a 
preference  for  free  oxygen,  are  not  readily  grown 
in  pour  plates,  it  is  often  advantageous  first  to 
pour  plates  of  suitable  media,  allow  them  to 
harden,  and  then  gently  smear  over  their  surfaces 
dilutions  of  the  infectious  material,  usually  in  three 
or  four  parallel  streaks.      (See  Fig.  31.) 

Upon  such  plates,  if  dilutions  have  been  prop- 
erly made,  and  this  is  only  a  question  of  judgment 
based  upon  an  estimation  of  the  numbers  of  bac- 
teria in  the  original  material,  discrete  colonies  of  the 
microorganisms  sought  for  may  develop,  and  can  be 
"fished"  in  the  usual  manner. 

The  media  most  favorable  for  the  cultivation  of 
various  microorganisms  will  be  discussed  in  the 
sections  dealing  "mth  the  individual  species. 

ANAEROBIC    METHODS 

We  have  seen  in  a  preceding  chapter    (p.  26) 

pjQ^  32^ Deep   ^^^^  many  bacteria,   the  so-called    anaerobes,  will 

Stae  Cultivation    develop  only  in   an  environment  from  which  free 
OF    Anaerobic   oxygen  has  been  excluded. 

Bacteria.  rpj^^  exclusion  of  oxygen  for  purposes  of  anaero- 

bic cultivation  may  be  accomplished  by  a  variety  of 
methods,  depending  upon  a  few  simple  principles  which  have  been 
applied,  either  separately  or  in  combination,  by  many  workers. 

The  earliest  methods  depended  upon  the  simple  exclusion  of  air  l^y 
mechanical  devices.  In  other  methods,  the  oxygen  of  the  air  is  displaced 
by  inert  gases  (hydrogen) ,  and  others  again  depend  upon  the  .oxygen- 
absorbing  qualities  of  alkaline  solutions  of  pyrogallol. 

Cultivation  by  the  Mechanical  Exclusion  of  Air. — Koch  succeeded  in 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA 


149 


growing  anaerobic  bacteria  upon  plates  by  simply  dropping  upon  the 
surface  of  the  inoculated  agar  or  gelatin  a  flat  piece  of  sterile  mica.  This 
method,  however,  rarely  succeeds  in  sufficiently  excluding  the  air. 

LiBORius'  Method.^ — This  method  consists  in  the  use  of  deeply  filled 
tubes  of  agar  or  gelatin,  from  which  all  oxygen  has  been  removed  by 
boiling  for  fifteen  minutes  or  more.     It  is  advantageous,  as  has  been 
pointed  out  in  the  section  on   anaerobiosis,   that 
media  used  for  this  purpose  should  contain  carbo- 
hydrates in  some  form,  perferably  glucose.     After 
boiling,  the  tubes  are  rapidly  transferred  to  ice 
water  so  that  as  little  oxygen  as  possible  may  be 
absorbed  during  the  hardening    of    the    medium. 
The  tubes  are  then  inoculated  by  deep  stabs.     After 
inoculation,  the  medium  may  be  covered  with  a 
thin  layer  of   agar,  gelatin,  or   oil    (albolin),   and 
further  sealed  with  sealing-wax  to  prevent  oxygen- 
absorption. 

This  method  may  be  utilized  for  the  isolation 
of  anaerobes  (as  in  the  original  method  of  Libo- 
rius)  by  inoculating  the  medium  just  before  it 
solidifies.  The  tubes  may  be  gently  shaken  in 
order  to  distribute  the  bacteria  throughout  the 
medium  and  then  rapidly  cooled.  In  this  case 
colonies  which  develop  may  be  scattered  through- 
out the  deeper  layers  of  the  agar  or  gelatin,  and 
may  be  "fished"  after  breaking  the  tube. 

Esmarch's  Method.2 — Von  Esmarch  has  applied 
the  principles  of  his  roll-tube  to  the  cultivation  of 
anaerobic  bacteria.  Gelatin  tubes  are  inoculated 
as  above  and  roll-tubes  prepared.  The  tubes  are 
then  set  into  cold  water  to  prevent  melting  of  the 
thin  gelatin  layer  and  the  interior  of  the  tube  is 
filled  with  melted  gelatin. 

Roux's  Method .2 — ^Anaerobic  bacteria  are  culti- 
vated by  sucking  the  inoculated  gelatin  or  agar  into  narrow  tubes, 
which  are  then  closed  at  both  ends  by  fusing  in  the  flame.     After 
growth  Tias  taken  place  the  tubes  are  broken  and  the  organism  re- 
covered by  "fishing." 

^  Idborius,  Zeit.  f .  Hyg.,  i,  1886.  2  y^^  Esmarch,  loc.  cit. 

3  Roux,  Ann.  Past.,  i,  1887. 


Fig,  33. — Deep 
Stab  Cultiva- 
tion OF  Anaero- 
bic Bacteria. 


150 


BIOLOGY  AND  TECHNIQUE 


Fluid  Media  Covered  with  Oil. — Erlenmeyer  flasks  or  othervessels 
are  partially  filled  with  glucose-bouillon  over  which  a  thin  layer  of  al- 
bolin  or  other  oil  is  allowed  to  flow.  The  oxygen  is  driven  out  of  the 
liquid  by  vigorous  boiling  for  fifteen  minutes  or  more. 

It  should  be  remembered  whenever  using  this  or  similar  methods  that 
a  layer  of  fluid  oil  does  not  form  an  impermeable  seal.     By  covering  an 

alkaline  pyrogallol  solution  with  oil  it 
can  easily  be  shown  that  oxygen  slowly 
diffuses  through  the  oil  into  the  medi- 
um below. 

The  simple  exclusion  of  air,  also, 
is  the  principle  underlying  the  culti- 
vation of  anaerobic  bacteria  in  the 
closed  arm  of  a  Smith  fermentation 
tube. 

Wright's  Method.' — Wright  has 
described  a  simple  and  excellent 
method  for  the  cultivation  of  anaero- 
bic bacteria  in  fluid  media.  The  ap- 
paratus necessary  is  easily  improvised 
with  the  materials  at  hand  in  any 
laboratory.  A  short  piece  of  glass 
tubing,  constricted  at  both  ends  and 
fitted  at  each  end  with  a  small  piece  of 
soft-rubber  tubing,  is  inserted  into  a 
test  tube  containing  nutrient  broth. 
The  upper  end  of  the  inserted  glass 
tubing  is  connected  by  the  rubber 
with  a  pipette  passed  through  the 
cotton  plug  in  the  tube.  The  entire 
apparatus,  plus  broth,  may  be  steril- 
ized after  being  put  together.  When 
a  cultivation  is  made,  the  fluid  in  the  test  tube  is  inoculated  as  usual.  The 
fluid  is  then  sucked  up  into  the  glass  tubing  until  this  is  completely  filled. 
A  downflow  of  the  fluid  is  then  prevented  by  placing  the  finger  over  the 
pipette  through  which  the  suction  has  been  made  or  by  constricting  a 
small  piece  of  rubber  tubing  attached  to  the  upper  end  of  the  pipette. 
The  entire  system  of  tubes  is  then  pushed  downward  in  such  a  way  that 
both  pieces  of  rubber  tubing,  attached  to  the  ends  of  the  httle  glass 

1  Wnght,  J.  H.    Quoted  from  Mallory  and  Wright,  "Path.  Technique,"  Phila.,  1904. 


Fig.  34.  —  Cultivation  op 
Anaerobes  in  Fluid  under 
Albolin. 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA 


151 


chamber,  are  kinked.  The  entire  apparatus  may  then  be  incubated. 
Growth  of  anaerobic  bacteria  takes  place  within  the  air-tight  chamber 
formed  by  the  short  glass  tubing  within  the  test  tube.  The  fluid  in 
the  test  tube,  outside  of  this  chamber,  usually  remains  clear. 

When  cultivation  has  been  successful,  the  bacteria  may  be  obtained 
either  for  morphological  study  or  for  further 
cultivation,  by  simply  allowing  the  fluid  to 
flow  out  of  the  little  air-tight  chamber  back 
into  the  test  tube.  The  method  is  simple  and 
usually  successful. 

Methods  Based  upon  the  Displacement  of  Air 
by  Hydrogen. — The  principle  of  air-displace- 
ment by  hydrogen,  first  utilized  by  Hauser,* 
has  been  widely  applied  to  the  cultivation  of 
anaerobic  bacteria.  In  substance  it  consists 
of  the  conduction  of  a  stream  of  hydrogen 
through  an  air-tight  chamber  in  which  plates 
or  tubes  containing  inoculated  media  have  been 
placed. 

For  the  production  of  hydrogen,  the  most 
convenient  apparatus  is  the  Kipp  hydrogen 
generator.  Hydrogen  is  generated  from  zinc 
and  sulphuric  acid  and  this  may  be  passed 
through  a  series  of  Woulfe-bottles,  containing 
solutions  of  lead  acetate  and  of  pyrogallic 
acid,  to  remove  traces  of  sulphuretted  hydro- 
gen and  of  oxygen,  respectively,  of  Lugol's 
solution  to  absorb  traces  of  acid  vapor,  and 
of  one  with  a  silver-nitrate  solution  to  take 
up  any  hydrogen  arsenide. 

For  the  preparation  of  anaerobic  condi- 
tions where  very  rigid  anaerobiosis  is  neces- 
sary, nitrogen  may  be  used,  which  can  be 
bought  in  tanks  from  commercial  firms. 

For     anaerobic     cultivation     upon     solid 
media,  the  inoculated  tubes  or  plates  are  placed  in  an  apparatus  such 
as  the  Novy  jar.     This  is  connected  with  the  hydrogen  apparatus  and 
hydrogen  allowed  to  flow  through  it  for  five  or  ten  minutes,  and  the 
stop-cocks  then  closed. 


Fig.  35.  —  Wright's 
Method  of  Anaerobic 
Cultivation  in  Fluid 
Media. 


^  Hauserj  "Ueber  Faulnissbakterien,"  1885. 


152 


BIOLOGY  AND  TECHNIQUE 


In  applying  the  hydrogen  method  to  fluid  media,  flasks  containing 
the  broth  are  fitted  with  sterile,  tightly  fitting  rubber  stoppers  per- 
forated by  two  holes,  through  which  glass  tubes  are  passed.  One  of 
these  tubes,  the  inlet,  passes  below  the  surface  of  the  liquid.  The  other 
one,  the  outlet,  extends  only  a  short  distance  below  the  stopper  and  is 
always  kept  above  the  surface  of  the  medium.  The  flasks  are  inoculated 
and  hydrogen  is  passed  through  the  medium  so  that  it  enters  the 
long  tube,  bubbles  up  through  the  fluid,  and  leaves  by  the  short  tube. 

The  broth  may  be  covered  with  a 
thin  layer  of  liquid  paraffin  or 
albolin. 

The  Use  ot  Pjnrogallic  Acid 
Dissolved  in  Alkaline  Solutions 
for  Oxygen  Absorption. — Buchner* 
has  applied  the  principle  of 
chemical  absorption  for  the  re^ 
moval  of  oxygen  to  the  cultiva- 
tion of  anaerobic  bacteria.  This 
has  been  made  use  of  in  a 
number  of  different  ways.  The 
method  is  based  upon  the  fact 
that  alkaline  solutions  of  pyro- 
gallol  possess  the  power  of  ab- 
sorbing large  quantities  of  free 
oxygen.  At  first  such  solutions 
are  of  a  light,  straw-color,  which 
becomes  dark  brown  as  oxygen 
is  absorbed.  The  absorption  of 
all  the  oxygen  in  the  environ- 
ment may  be  assumed  when 
there  is  no  further  deepening  of 
the  brown  color. 

Buchner  first  utilized  this 
principle  by  placing  a  small  wire  or  glass  holder  within  a  large  test 
tube,  dropping  dry  pyrogallol  (pyrogallic  acid)  into  the  bottom  of  this 
tube,  then  running  thirty  per  cent  sodium  hydrate  solution  into  it, 
and  inserting  within  this  large  tube  a  smaller  test  tube  containing  the 
inoculated  culture  medium.      The  large  tube  was  then  tightly  closed 


Fig    36.— Novy  Jar. 


1  Buchner,  Cent.  f.  Bakt.,  I,  iv,  1888. 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA 


153 


with   a  rubber  stopper.     In  this  way,  the  air  space  surrounding  the 
smaller  tube  was  rendered  oxygen  free. 

A  simple  modification  of  the  preceding  method  of  Buchner  has 
been  devised  by  Wright.^  Stab-cultures  of  gelatin  or  agar  in  test 
tubes  are  made  in  the  usual  way.  The  cotton  stopper  closing  the  tube 
is  then  thrust  into  the  tube  to  such  a  depth  that  its  upper  end  lies  at 
least  1  cm.  below  the  mouth  of  the  tube. 
A  small  quantity  of  sodium  or  potassium 
hydrate  solution  in  which  some  pyrogallic 
acid  has  been  dissolved,  is  then  allowed  to 
flow  on  to  the  cotton  of  the  plug  and  the 
mouth  of  the  tube  is  immediately  sealed  by 
a  tightly  fitting  rubber  stopper.  The  cotton 
stopper  in  these  cases  must  be  made  of  ab- 
sorbent cotton;  1.5  to  2.5  c.c.  of  the  pyro- 
gallicr  acid  solution  is  usually  sufl^icient  for 
test  tubes  of  ordinary  size. 

For  cultivation  of  anaerobic  bacteria 
upon  agar  slants,  a  simple  technique  may  be 
appHed  and  easily  improvised  in  the  labora- 
tory as  follows:  The  tube  of  slant  agar  is 
inoculated  with  the  infectious  material  in  the 
usual  way.  It  is  then,  with  stopper  removed, 
inverted  into  a  tumbler  or  beaker  containing 
about  a  gram  of  dry  pyrogallic  acid.  A 
small  quantity  of  a  five  per  cent  or  three 
per  cent  sodium  hydrate  solution  is  then  run 
into  the  tumbler  and  this  is  covered  with  a 
thin  layer  of  liquid  paraffin  or  albolin  before 
the  pyrogallic  acid  has  been  completely  dis- 
solved. In  this  way,  completely  anaerobic 
conditions  are  obtained  in  the  tube  and  the 
growth  of  anaerobes  takes  place  upon  the 
surface  of  the  slant. 

For  the  cultivation  of  anaerobes  in  Petri 
dishes,  for  purposes    of  separation,  a  combination   of  the   pyrogallic 
acid  method    and   the   hydrogen  displacement  methods  is  often  em- 
ployed.    For  this  purpose  the  jars  devised  by  Novy  and   by  Bulloch 
are  extremely  convenient. 

1  Wright,  Jour,  of  the  Boston  Soc,  of  Med.  Sci,,  Dec,  1900, 


k. 


Fig.  37, — Wright's  Meth- 
od OF  Anaerobic  Cultiva- 
tion BY  THE  Use  of  Pyro- 
gallic Acid  Solution. 


154 


BIOLOGY  AND  TECHNIQUE 


In  using  the  Novy  jar,  the  inoculated  plates  are  set  upon  a  wire 
frame  resting  about  an  inch  above  the  bottom  of  the  jar.  The  cover 
is  then  tightly  set  in  place  and  the  air  in  the  jar  exhausted  by  means  of  a 
suction  pump.  The  arrangement  of  the  double  stop-cock  in  the  top 
renders  it  possible  now,  by  simply  turning  this,  to  admit  hydrogen  from 
a  Kipp  generator  into  the  jar.    The  process  of  alternate  exhaustion  and 

admission  of   hydrogen   may  be 
several  times  repeated. 

A  combination  of  air  exhaus- 
tion, oxygen  absorption,  and  hy- 
drogen replacement  may  be  prac- 
ticed in  jars  such  as  that  shown 
in  Fig.  39.  Tubes  or  plates  after 
inoculation  are  placed  in  this  jar, 
on  a  raised  wire  frame.  Dry  py- 
rogallic  acid  is  placed  in  the 
bottom  of  the  jar  and  the  cover 
tightly  fitted.  An  opening  in 
the  side  of  the  jar  connects  its 
interior  with  a  bottle  containing 
sodium  or  potassium  hydrate  so- 
lution. Through  the  stopper  of 
this  bottle  pass  two  glass  tubes, 
one  of  them  of  such  length  that 
it  can  be  pushed  down  into  the 
alkaline  solution,  or  pulled  up- 
ward above  the  level  of  the  fluid. 
This  tube  connects  the  jar  with 
the  bottle.  The  other  glass  tube 
is  short,  passing  just  through  the 
stopper  and  at  the  top  made  in 
the  form  of  a  T,  one  arm  of  the  T 
being  connected  with  a  Kipp  hydrogen  generator,  the  other  with  a 
suction-pump. 

After  the  jar  has  been  sealed,  the  glass  tube  connecting  the  jar  and 
the  bottle  is  raised  above  the  level  of  the  fluid  in  the  bottle  and,  the  con- 
nection to  the  hydrogen  generator  being  closed,  the  air  in  the  jar  is 
exhausted  with  the  suction-pump.  Connection  to  the  suction  may  then 
be  closed,  and  the  other  arm  of  the  T  being  open,  hydrogen  is  allowed  to 
flow  into  the  jar.    Alternate  suction  and  hydrogen  replacement  may  be 


Fig.  38. 


-Jar  for  Anaerobic  Cul- 
tivation. 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA 


155 


carried  out  two  or  th/ee  times.  After  the  last  exhaustion,  the  glass  tube 
in  the  bottle  connecting  it  with  the  jar  is  pushed  down  into  the  fluid, 
and  the  vacuum  will  draw  the  sodium  hydrate  solution  into  the  bottom 
of  the  jar,  dissolving  the  pyrogallol,  which  will  then  absorb  any  traces  of 
free  oxygen  remaining  in  the  jar.  Hydrogen  is  again  introduced  and  tlie 
jar  closed.  If  exhaustion  of  oxygen  has  been  sufficiently  thorough, 
the  pyrogallic  solution  in  the  bottom  of  the  jar  will  remain  light  brown. 

A  simple  method  for  the  separation  of  anaerobes  in  plates  without  the 
use  of  hydrogen  or  of  specially  constructed  jars,  may  be  carried  out  as 
follows  ^:  The  apparatus  used  consists  of  two  circular  glass  dishes,  fitting 
one  into  the  other  as  do  the  halves  of  a  Petri  dish,  and  similar  to  these 
in  every  respect  except  that  they  are  higher,  and  that  a  slightly  greater 
space  is  left  bet^i^een  their  sides  when  they  are  placed  together.     The 


From  hydrogen 
generator 

To  exhaustion 
pump 


Fig.  39. — Apparatus  for  Combining  the  Methods  of  Exhaustion,  Hydrogen, 
Replacement,  and  Oxygen  Absorption. 


dishes  should  be  about  three-fourths  to  one  inch  in  height,  they  need 
be  of  no  particular  diameter,  although  those  of  about  the  same  size  as 
the  usual  Petri  dish  are  most  convenient.  An  important  requirement 
necessary  for  the  success  of  the  method  is  that  the  trough  left  between  the 
two  plates,  when  put  together,  shall  not  be  too  broad,  a  quarter  of  an 
inch  being  the.  most  favorable. 

Into  the  smaller  of  these  plates  the  inoculated  agar  is  poured  exactly 
as  this  is  done  into  a  Petri  dish  in  the  ordinary  aerobic  work.  Pro- 
longed boiling  of  the  agar  before  plating  is  not  essential.  When  the  agar 
film  has  become  sufficiently  hard  on  the  bottom  of  the  smaller  dish,  the 
entire  apparatus  is  inverted.     The  smaller  dish  is  now  lifted  out  of  the 


1  Zinsser,  Jour.  Exp.  Med.,  viii,  1906. 


156 


BIOLOGY  AND  TECHNIQUE 


larger,  and  placed,  still  inverted,  over  a  moist  surface — a  towel  or  the 
wet  surface  of  the  table — to  prevent  contamination.  Into  the  bottom 
of  the  larger  dish,  which  now  stands  open,  there  is  placed  a  quantity 
(about  3  grams)  of  dry  pyrogallic  acid.  Into  this,  over  the  pyrogallic 
acid,  the  smaller  dish,  still  inverted,  is  then  placed.  A  five  per  cent  solu- 
tion of  sodium  hydrate  is  poured  into  the  space  left  between  the  sides  of 
the  two  dishes,  in  quantity  sufficient  to  fill  the  receiving  dish  one-half 
full.  While  this  is  gradually  dissolving  the  pyrogallic  acid,  albolin, 
or  any  other  oil  (and  this  is  the  only  step  that  requires  speed),  is 
dropped  from  a  pipette,  previously  filled  and  placed  in  readiness,  into 


Fig.  40. — Simple  Apparatus  for  Plate    CuLTr7ATioN  of  Anaerobic  Bacteria. 

(Zinsser.) 

the  same  space,  thus  completely  sealing  the  chamber  formed  by  the  two 
dishes. 

If  these  steps  have  been  performed  successfully,  the  pyrogallic  solu- 
tion will  at  this  time  appear  of  a  light  brown  color,  and  the  smaller  plate, 
with  its  agar  film,  will  float  unsteadily  above  the  other.  Very  rapidly, 
as  the  pyrogallic  acid  absorbs  the  free  oxygen  in  the  chamber,  this  plate 
is  drawn  down  close  to  the  other,  and  the  acid  assumes  a  darker  hue, 
which  remains  without  further  deepening  even  after  three  or  four  days' 
incubation. 

The  Use  of  Fresh  Sterile  Tissue  as  an  Aid  to  Anaerobic  Cultivation. 
— The  addition  of  small  pieces  of  fresh  sterile  tissue  (rabbit  or  guinea- 
pig)  to  culture  tubes,  either  solid  or  fluid,  greatly  favors  the  growth  of 
anaerobic  bacteria.  By  such  a  method  anaerobes  can  be  made  to  de- 
velop even  when  other  precautions  for  the  establishment  of  anaerobiosis 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA 


157 


are  imperfectly  observed.  This  was  noticed  first  by  Theobald  Smith  and 
by  Tarozzi  and  has  become  an  extremely  useful  reenforcement  to  other 
methods.     It  has  been  utilized  most  extensively  by  Noguchi  of  recent 


^ 

,  jS, 

Fig.  41. — Incubator. 


years  in  his  technique  for  the  cultivation  of  various  treponemata. 
The  simplest  way  to  apply  this  method  is  to  place  a  piece  of  freshly 
excised  rabbit  kidney,  testicle,  or  spleen  into  the  bottom  of  a  high  test 
tube  (20  cm.)  and  then  pouring  over  it  the  culture  fluid.     Kidney  or 


158 


BIOLOGY  AND  TECHNIQUE 


other  tissues  are  more  suitable  for  this  purpose  than  Uver  tissue  since 
the  latter  is  not  easily  obtained  in  a  sterile  condition,  bacteria 
often  getting  into  it  during  life  through  the  portal  circulation.  The 
action    of    the    tissues  depends    probably   upon   its   great   reducing 


power. 


THE  INCUBATION  OF  CULTURES 

After  bacteria  have  been  transferred  to  suitable  culture  media,  it 
is  necessary  to  expose  them  to  a  temperature  favorable  to  their  develop- 
ment. In  the  case  of  many  saprophytes, 
the  ordinary  room  temperature  is  suffi- 
ciently near  the  optimum  to  obviate  the 
use  of  any  special  apparatus  for  maintain- 
ing a  suitable  temperature;  in  the  case 
of  most  pathogenic  bacteria,  however, 
the  body  temperature  of  man,  37.5° 
C,  is  either  a  necessary  requirement 
for  their  growth,  or  at  any  rate 
favors  speedy  and  characteristic  develop- 
ment. 

For  the  purpose  of  obtaining  a  uniform 
temperature  of  any  required  degree,  the 
apparatus  in  general  use  is  the  so-called 
incubator  oi:  thermostat.  This  may  be 
adjusted  for  gelatin  cultivation  at  20  to 
22°  C,  or  for  agar,  broth,  or  other  media 
at  37.5°  C. 

Incubators,  while  varying  in  detail, 
are  all  constructed  upon  the  same  prin- 
ciples. They  consist  of  double-walled  copper  chambers,  which  are 
fitted  with  a  set  of  double  doors,  the  outer  being  made  of  asbestos- 
covered  metal,  the  inner  of  glass.  (See  Fig.  41.)  The  space  be- 
tween the  two  walls  is  filled  with  water,  which,  being  a  poor 
heat  conductor,  tends  to  prevent  rapid  changes  of  temperature 
within  the  chamber  as  the  result,  of  changes  in  the  external 
environment.  Both  walls  are  perforated  above  by  openings  to 
admit  thermometers  into  the  interior  and  one  wall  is  perforated 
so  that  a  thermo-regulator  may  be  inserted  into  the  water 
jacket.      The   under   surface    of   the   chamber   is    heated    by  a  gas 


Fig.  42. 


Fig.  43. 


Fig.  42. — Thermo-regulator 
(Lautenschlagef.) 


Fig.  43.- 


-Thermo-regulator. 
(Reichert.) 


METHODS    USED    IN    CULTIVATION    OF    BACTERIA  159 

flame,  the  size  of  which  is  automatically  regulated  by  the  thermo- 
regulator. 

A  number  of  thermo-regulators  are  on  the  market,  all  of  them  con- 
structed upon  modifications  of  the  same  principle.  One  of  the  most 
efficient  of  those  in  common  use  is  that  shown  in  Fig.  42.  This  con- 
sists of  a  long  tube  of  glass  fitted  with  a  metal  cap  through  which  an  in- 
let tube  (A)  projects  into  the  interior.  Slightly  below  the  middle  of  the 
tube  there  is  a  glass  diaphragm  separating  its  interior  into  two  com- 
partments. In  the  middle  of  the  diaphragm  an  aperture  leads  into  a 
spiral  of  glass  which  projects  into  the  lower  compartment.  The  lower 
compartment  is  filled  with  ether  and  mercury.  The  lower  end  of  the  inlet 
tube  (A)  has  a  wedge-shaped  slit.  The  gas  from  the  supply  pipe 
passing  through  the  tube  (A)  is  conducted  through  the  slit-like  opening 
in  its  lower  end  into  the  inner  chamber  and  passes  out  to  the  burner 
through  the  elbow  (B).  When  the  temperature  is  raised,  the  ether  and 
mercur}^  in  the  lower  chamber  expand  and  the  n:ercury  rises  in  the 
upper  chamber,  gradually  restric  ting  the  opening  through  the  V-shaped 
slit  •  in  the  inlet  tube.  Thus  the  gas  supplied  to  the  burner  is 
diminished,, the  flame  reduced,  and  the  temperature  again  falls.  The 
temperature  can  be  arbitrarily  adjusted  by  raising  or  lowering 
the  inlet  tube.  A  scale  at  the  upper  ,end  of  the  inlet  tube  allows 
exact  adjustment.  Complete  shutting  off  of  the  gas  is  prevented  by  a 
small  circular  opening  placed  in  the  inlet  tube  just  above  the  slit. 

Another  cheaper  and  simpler  thern  o-regulator  is  shown  in 
Fig.  43.  This  consists  of  a  long  tube  -open  at  the  top  and  fitted 
about  1^  inches  fro'm.  the  top  with  two  hollow  glass  elbows.  One  of  these 
elbows  remains  open,  the  other,  situated  on  a  slightly  lower  level,  is  closed 
by  a  brass  screw-cap.  The  tube  is  filled  with  mercury  to  a  point  slightly 
above  the  level  of  the  elbow  containing  the  screw-cap.  The  height  of 
the  mercury  can  thus  be  increased  or  decreased  by  screwing  in  or  out 
upon  the  cap.  Into  the  upper  end  of  the  tube  there  is  fitted  another 
device  which  consists  of  a  T-shaped  system  of  glass  tubes,  one  arm 
of  the  T  being  open  and  the  other  closed,  the  perperdicular  leg  of  the 
T  tapering  to  a  minute  opening  at  the  bottom.  The  gas  passes  into 
one  arm  of  the  T  down  through  the  tapering  leg  and  into  the  space 
immediately  above  the  mercury.  It  then  passes  out  through  the  open 
elbow  of  the  main  tube.  As  the  mercury  rises,  it  gradually  diminishes 
the  space  between  its  surface  and  the  small  opening  in  the  tapering  tube 
above  it,  finally  completely  shutting  off  the  gas  from  this  source.  Gas  can 
now  pass  only  through  a  minute  hole  perforating  the  vertical  leg  of  the 
12 


160 


BIOLOGY  AND  TECHNIQUE 


T  an  inch  above  its  end.     The  flame  decreases  and  the  temperature 
again  sinks.  . 

Since  gas  pressure  in  laboratories  is  apt  to  vary,  it  is  convenient  to 
interpose  between  the  gas  supply  and  thermo-regulator  some  one  of  the 
various  forms  of  gas-pressure  regulators.  The  use  of  these  is  not  ab- 
solutely necessary  but  aids  considerably  in  the  maintenance  of  a  con- 
stant temperature.  The  one  most  commonly  employed  is  the  so-called 
Moitessier  apparatus.  This  consists  of  a  cylindrical  metal  chamber 
within  which  there  is  fitted  an  inverted  metal  bell.  Glycerin  is  poured 
into  the  cylinder  to  the  depth  of  about  two  inches.    An  inlet  pipe  con- 


FiG.  44. — Moitessier  Gas-Pressure  Regulator. 


ducts  gas  into  the  open  space  between  the  top  of  the  glycerin  and  the  bell. 
From  the  top  of  the  bell  is  suspended  a  conical  piece  of  metal  which  hangs 
free  in  the  outlet  pipe.  As  the  gas  pressure  under  the  bell  increases, 
this  is  raised  and  the  opening  of  the  outlet  pipe  is  gradually  diminished 
by  the  cone.  Thus  the  relation  between  the  pressure  in  the  inlet  pipe 
and  the  actual  quantity  of  gas  passing  through  is  equalized.  A  cup  con- 
nected to  the  top  of  the  bell  through  the  roof  of  the  cylinder  by  a  bar  can 
be  filled  with  birdshot  and  the  pressure  against  the  gas  can  thus  be 
modified  to  conform  with  existing  conditions. 


METHODS    trsEr)    m    CUitiVAtlON    01?    BACTERIA 


161 


Colony  Study. — Cultures  are  usually  incubated  for  from  twelve  to 
forty-eight  hours.  Considerable  aid  to  the  recognition  of  species  ia 
derived  from  the  observation  of  both  the  speed  of  growth  andjbhe  ap- 
pearance of  the  colonies.  It  is  therefore  necessary  to  proceed  in  the 
study  of  developed  co  onies  in  a  systematic  way.  The  development  of 
colonies  should  be  observed  in  all  cases  both  upon  gelatin  and  upon  agar. 
In  forming  any  judgment  about  colonies,  the  acidity  or  alkalinity,  and 
the  special  constitution  of  the  media  should  be  taken  into  account. 
The  colonies  are  carefully  examined  with  a  hand  lens  and  with  the  low 
power  (Leitz  No.  2,  Zeiss  AA,  Ocular  No.  2)  of  the  microscope.  The 
colonies  should  be  observed  as  to  size,  outline,  transparency,  texture, 
color,  and  elevation  from  the  surface  of  the  media.    Much  information, 


Fig.  45. — Variations  in   the   Conformation  of  the  Borders   of   Bacterial 
Colonies.    (After  Chester.) 


also,  can  be  obtained  by  observing  .whether  a  colony  appears  dry, 
mucoid,  or  glistening,  like  a  drop  of  moisture.  By  a  careful  obser- 
vation of  these  points,  definite  differentiation,  of  course,  can  not  usu- 
ally be  made,  but  much  corroborative  evidence  can  be  obtained  which 
may  guide  us  in  the  methods  to  be  adopted  for  further  identification 
and  for  a  final  summing  up  of  species  characteristic  as  a  whole. 

The  Counting  of  Bacteria. — It  is  often  necessary  to  determine  the 
number  of  bacteria  per  c.c.  contained  in  water,  milk,  or  other  substances. 
For  this  purpose  definite  quantities  of  the  material  to  be  analyzed  are 
mixed  with  gelatin  or  agar  and  poured  into  Petri  plates.  The  exact 
dilutions  of  the  suspected  material  must  largely  depend  upon  the  number 
of  germs  which  one  expects  to  find  in  it.  The  plates,  if  prepared  with 
gelatin,  are  allowed  to  develop  at  room  temperature  for  twenty-four  to 


162 


BIOLOGY  AND  TECHNIQUE 


forty-eight  hours.  If  agar  has  been  used,  they  are  usually  placed  in  the 
incubator  at  37.5°  C.  At  the  end  of  this  time,  the  colonies  which  have 
developed  are  enumerated.  For  this  purpose,  a  Petri  dish  is  placed  upon  a 
Wolffhugel  plate.  This  plate  consists  of  a  disk  or  square  of  glass  which  is 
divided  into  small  squares  of  one  square  centimeter  each.  Diagonal  lines 
of  these  sq\iares  running  at  right  angles  to  each  other  are  subdivided  into 
nine  divisions  each  in  order  to  facilitate  counting  when  the  colonies  are 
unusually  abundant.  The  Petri  dish  is  placed  upon  the  plate  in  such  a 
way  that  the  center  of  the  dish  corresponds  to  the  center  of  the  plate. 


Fig.  46. — WolffhugeIj  Counting  Plate. 


The  colonies  in  a  definite  number  of  squares  are  then  counted.  The 
greater  the  number  of  squares  that  are  counted  the  more  accurate  the 
estimation  will  be.  When  the  growth  is  so  abundant  that  only  a  limited 
number  of  squares  can  be  counted,  these  should  be  chosen  as  much  as 
possible  from  different  parts  of  the  plate,  and  in  practice  one  counts 
usually  six  squares  in  one  direction  and  six  at  right  angles  to  these,  so 
as  to  preclude  errors  arising  from  unequal  distribution.  The  final  calcu- 
lation is  then  made  by  ascertaining  the  average  number  of  colonies  con- 
tained in  each  square  centimeter.    If  standard  Petri  dishes  have  been 


METHODS  USED  IN  CULTIVATION  OF  BACTERIA  163 

used,  this  is  multiplied  by  63.6,  the  number  of  squares  in  the  area  of  the 
dish,  and  then  by  the  dilution  originally  used. 

Thus  if  twelve  squares  have  been  counted  with  a  total  number  of 
one  hundred  and  forty-four  colonies — the  average  for  each  square  is 
twelve.  Twelve  times  63.6  is  763.2,  which  represents  the  total  number 
of  colonies  in  the  plate.  Now  if  0.1  c.c.  of  the  original  material 
(water  or  milk)  has  been  plated,  this  material  may  be  assumed  to  have 
contained  10  X  763.2,  or  7,632  bacteria  to  each  cubic  centimeter. 

If  dishes  of  an  unusual  size  are  employed,  the  square  area  must  be 
ascertained  by  measuring  the  radius  and  multiplying  its  square  by  n 
(TT  X  R2  =  area)  ( 7:=  3.141592). 


CHAPTER  IX 

METHODS  OF  DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERU 
ANIMAL  EXPERIMENTATION 

Gas  Formation. — Bacteria  of  many  varieties  produce  gas  from  the 
proteid  and  the  carbohydrate  constituents  of  their  environment. 

Gas  formation  can  be  observed  in  a  very  simple  manner  by  making 
stab  cultures  in  gelatin  or  agar  containing  the  fermentable  nutrient 
substances.  In  such  cultures  bubbles  of  gas  will  form  along  the  track 
of  the  inoculation,  or,  in  the  case  of  such  semisolid  media  as  the  tube 
medium  of  Hiss,  will  spread  throughout  the  tube.  In  the  case  of  some 
anaerobes  gas  formation  in  stab  cultures  will  occur  to  such  an  extent  that 
the  medium  v/ill  split  and  break.  It  should  be  borne  in  mind  in  carrying 
out  such  methods  that  air  is  readily  carried  into  the  medium  with 
the  inoculating  needle  or  loop  by  splitting  of  the  medium,  also  that 
media  which  have  been  stored  in  the  cold  may  absorb  air.  Expansion 
of  the  air  in  such  tubes  may  simulate  small  amounts  of  gas  formation 
and  lead  to  error.  It  is  advisable,  therefore,  whenever  making  stab 
inoculations  with  the  above  purpose,  to  heat  the  media  and  rapidly 
cool  them  before  use. 

A  more  accurate  method  of  gas  determination  is  by  the  use  of  fer- 
mentation tubes,  such  as  those  devised  by  Smith.  The  gas  which  is 
formed  collects  in  the  closed  arm  of  the  fermentation  tube  and  may  be 
quantitatively  estimated.  The  fermentation,  with  gas  production,  of 
certain  substances  such  as  carbohydrates,  may  be  determined  by  adding 
these  materials  in  a  pure  state  to  the  media  before  inoculation  with 
organisms. 

In  the  case  of  carbohydrates  this  method  has  proved  of  great  differ- 
ential value,  since  the  power  of  splitting  specific  carbohydrates  with  gas 
production  is  a  species  characteristic  of  great  constancy  for  many  forms 
of  bacteria. 

Analysis  of  Gas  Formed  by  Bacteria. — Carbon  Dioxide. — For 
the  estimation  both  qualitatively  and  roughly  quantitatively  of  carbon 
dioxide  produced  by  bacteria,  cultures  are  grown  in  fermentation  tubes 
containing  sugar-free  broth  (see  page  125)  to  which  one  per  cent  of  pure 
dextrose,  lactose,  saccharose,  or  other  sugars  has  been  added.  The  tubes 
are  incubated  until  the  column  of  gas  formed  in  the  closed  arm  no  longer 

X64 


DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERU        165 

increases  <,cwenty-four  to  forty-eight  hours).  The  level  of  the  fluid 
b  the  closed  arm  is  then  accurately  marked  and  the  column  of  gas 
Daeasured. 

The  bulb  of  the  fermentation  tube  is  then  completely  filled  with 
y  NaOH  solution,  the  mouth  closed  with  a  clean  rubber  stopper,  and 
the  bulb  inverted  several  times  in  order  to  mix  the  gas  with  the  soda 
solution.  The  tube  is  then  again  placed  in  the  upright  position,  allow- 
ing the  gas  remaining  to  collect  in  the  closed  arm.  The  gas  lost  may 
be  roughly  estimated  as  consisting  of  CO  2. 

Hydrogen. — The  gas  remaining,  after  removal  of  the  CO  2  in  the  pre- 
ceding experiment,  at  least  when  working  with  carbohydrate  solutions, 


Fig.  47. — ^Types  op  Fermentation  Tubes. 


may  be  estimated  as  hydrogen.  When  allowed  to  collect  near  the  mouth, 
further  evidence  of  its  being  hydrogen  may  be  gained  by  exploding  it 
with  a  lighted  match. 

Hydrogen  Sulphide  (H2S,  Sulphuretted  hydrogen). — In  alkaline 
media  sulphuretted  hydrogen,  if  formed,  will  not  collect  as  gas,  but 
will  form  a  sulphide  with  any  alkali  in  the  solution.  For  the  estimation 
of  the  formation  of  hydrogen  sulphide,  bacteria  are  cultivated  in  a  strong 
pepton  solution  to  which  0.1  c.c.  of  a  one  per  cent  solution  of  ferric 
tartrate  or  lead  acetate  has  been  added.  The  addition  of  these  substances 
gives  rise  to  a  yellowish  precipitate  in  the  bottom  of  the  tubes.    If,  on 


166  BIOLOGY  AND  TECHNIQUE 

subsequent  inoculation,  the  bacteria  produce  Ho  S,  this  precipitate  will 
turn  black.  The  solution  recommended  by  Pake  for  this  test  is  prepared 
as  follows: 

1.  Weigh  out  30  grams  of  pepton  and  emulsify  in  200  c.c.  of  tap  water  at  60°  0. 

2.  Wash  into  a  liter  flask  with  80  c.c.  tap  water. 

3.  Add  sodium  chloride  5  grams  and  sodium  phosphate  3  grams. 

4.  Heat  at  100°  C.  for  30  minutes,  to  dissolve  pepton. 

5.  Filter  through  paper. 

6.  Fill  into  tubes,  10  c.c.  each,  and  to  each  tube  add  0.1  c.c.  of  a  one  per  cent 
solution  of  ferric  tartrate  or  lead  acetate.    These  solutions  should  be  neutral. 

7.  Sterilize.* 

Accurate  quantitative  gas  analyses  of  bacterial  cultures  can  be 
made  only  by  the  more  complicated  methods  used  in  chemical  labora- 
tories for  quantitative  gas  analysis.  The  gas,  in  such  cases,  is  collected 
in  a  bell  jar  mounted  over  mercury,  and  subjected  to  analysis  by  the 
usual  method  described  in  works  on  analytical  chemistry. 

Acid  and  Alkali  Formation  by  Bacteria. — Many  bacteria  produce  acid 
or  alkaline  reactions  in  culture  media,  their  activity  in  this  respect 
depending  to  a  large  extent  upon  the  nature  of  the  nutrient  material. 
Many  organisms  which  on  carbohydrate  media  produce  acid  will  give 
rise  to  alkali  if  cultivated  upon  media  containing  only  proteids. 

Information  as  to  the  production  of  acid  or  alkali  can  be  obtained 
by  the  addition  of  one  of  a  variety  of  indicators  to  neutral  media.  The 
indicators  most  often  employed  for  this  purpose  are  litmus  or  neutral 
red.  Changes  in  the  color  of  these  indicators  show  whether  acids  or 
alkalis  have  been  produced. 

Great  help  in  differentiation  is  obtained  by  adding  chemically  pure 
carbohydrates  to  media  to  which  litmus  has  been  added  and  then  de- 
termining whether  or  not  acid  is  formed  from  the  substances  by  the 
microorganisms.  These  tests  have  been  of  special  importance  in  the 
differentiation  of  the  typhoid-colon  groups  of  bacilli. 

Quantitative  estimation  of  the  degree  of  acidity  or  alkalinity  pro- 
duced by  bacteria  may  be  made  by  careful  titration  of  definite  volumes 
of  the  medium  before  and  after  bacterial  growth  has  taken  place. 

The  variety  of  acid  formed  by  bacteria  depends  largely  upon  the 
nature  of  the  nutrient  medium.  The  acids  most  commonly  resulting 
from  bacterial  growth  are  lactic,  acetic,  oxalic,  formic,  and  hippuric 
acids.  Qualitative  and  quantitative  estimation  of  these  acids  may  be 
made  by  any  of  the  methods  employed  by  analytical  chemists. 

*  Quoted  from  Eyre,  "  Bact.  Technique/'  Phila.,  1903. 


DETERMINING  BIOLOGICAL  ACTIVITIES  OF  RACTERIA        167 

Indol  Production  by  Bacteria. — Many  bacteria  possess  the  power  of 
producing  indol.  Though  formerly  regarded  as  a  regular  accompani- 
ment of  proteid  decomposition,  later  researches  have  shown  that  indol 
production  is  not  always  coexistent  with  putrefaction  processes  and 
occurs  only  when  pepton  is  present  in  the  pabulum. 

Indol  formation  by  bacteria  is  determined  by  the  so-called  nitroso- 
indol  reaction.  Organisms  are  grown  in  sugar-free  pepton  broth  or  in  the 
pepton-salt  bouillon  of  Dunham.  (See  page  126.)  Media  containing 
fermentable  substaijces  are  not  favorable  for  indol  production  since  acids 
interfere  with  its  formation.  The  cultures  are  usually  incubated  for  three 
or  four  days  at  37°  C.  At  the  end  of  this  time,  ten  drops  of  con- 
centrated sulphuric  acid  are  run  into  each  tube.  If  a  pink  color 
appears,  indol  is  present,  and  we  gather  the  additional  information 
that  the  microorganism  in  question  has  been  able  to  form  nitrites 
by  reduction  (e.g.,  cholera  spirillum).  If  the  pink  color  does  not 
appear  after  the  addition  of  the  sulphuric  acid  alone,  nitrites  must 
be  supplied.  This  is  done  by  adding  to  the  fluid  about  1  c.c.  of  a  0.01 
per  cent  aqueous  solution  of  sodium  nitrite.  The  sodium  nitrite  solu- 
tion does  not  keep  for  any  length  of  time  and  should  be  freshly  made  up 
at  short  intervals. 

Phenol  Production  by  Bacteria. — Phenol  is  often  a  by-product  in  the 
course  of  proteid  cleavage  by  bacteria.  To  determine  its-  presence  in 
cultures,  bacteria  are  cultivated  in  flasks  containing  about  50-100  c.c. 
of  nutrient  broth.  After  three  to  four  days'  growth  at  37°  C,  5  c.c.  of 
concentrated  HCl  are  added  to  the  culture,  the  flask  is  connected  with  a 
condenser,  and  about  10-20  c.c.  are  distilled  over. 

To  the  distillate  may  be  added  0.5  c.c.  of  Millon's  reagent  (solution  of 
mercurous  nitrate  in  nitric  acid),  when  a  red  color  will  indicate  phenol; 
or  0.5  c.c.  of  a  ferric  chloride  solution,  which  will  give  a  violet  color  if 
phenol  is  present. 

Reducing  Powers  of  Bacteria. — The  power  of  reduction,  possessed  by 
many  bacteria,  is  shown  by  their  ability  to  form  nitrites  from  nitrates. 
This  is  easily  demonstrated  by  growing  bacteria  upon  nitrate  broth 
(see  page  126).  Bacteria  are  transferred  to  test  tubes  containing  this 
solution  and  allowed  to  grow  in  the  incubator  for  four  or  five  days. 
The  presence  of  nitrites  is  then  chemically  determined.^ 

^  We  are  indebted  to  Dr.  J.  P.  Mitchell,  of  Stanford  University,  for  the  following 
technique  for  nitrite  tests: 

I.  SulphauiUc  Acid. — Dissolve  0.5  g.  in  150  c.c.  of  acetic  acid  of  Sp.  Gr.  1.04. 


168  BIOLOGY  AND  TECHNIQUE 

In  bacteriological  work  4  c.c.  of  the  culture  fluid  is  poured  into  a 
clean  test  tube,  and  to  it  are  gradually  added  2  c.c.  of  the  mixed  test 
solutions.  A  pink  color  indicates  the  presence  of  nitrites,  the  intensity 
of  the  color  being  proportionate  to  the  amount  of  nitrite  present. 

The  reducing  powers  of  bacteria  may  also  be  shown  by  their  ability 
to  decolorize  litmus,  methylene-blue,  and  some  other  anilin  dyes, 
which  on  abstraction  of  oxygen  form  colorless  leukobases. 

Enzyme  Action. — The  action  of  the  enzymes  produced  by  bacteria 
may  be  demonstrated  by  bringing  the  bacteria,  or  their  isolated  fer- 
ments, into  contact  with  the  proper  substances  and  observing  both  the 
physical  and  chemical  changes  produced.  In  obtaining  enzymes  free 
from  living  bacteria,  it  is  convenient  to  kill  the  cultures  by  the  addition 
either  of  toluol  or  of  chloroform.  Both  of  these  substances  will 
destroy  the  bacteria  without  injuring  the  enzymes.  Enzymes  may  also 
be  obtained  separate  from  the  bodies  of  the  bacteria  by  filtration. 

Proteolytic  Enzymes. — The  most  common  evidences  of  proteolytic 
enzyme  action  observed  in  bacteriology  are  the  liquefaction  of  gelatin, 
fibrin  or  coagulated  blood-serum,  and  the  peptonization  of  milk.  This 
may  be  observed  both  by  allowing  the  proper  bacteria  to  grow  upon 
these  media,  or  by  mixing  sterilized  cultures  with  small  quantities  of 
these  substances.^  The  products  of  such  a  reaction  may  be  separated 
from  the  bacteria  by  filtration  and  then  tested  for  pepton  by  the  biuret 
reaction. 

Proteolytic  ^  enzymes  may  also  be  determined  by  growing  the  bac- 
teria upon  fluid  media  containing  albumin  solutions,  blood  serum,  or 
milk  serum,  then  precipitating  the  proteids  by  the  addition  of  ammonium 
sulphate  (about  30  grams  to. 20  c.c.  of  the  culture  fluid)  and  warming 
between  50  to  60°  C.  for  thirty  minutes.  The  precipitate  is  then  filtered 
off,  the  filtrate  made  strongly  alkaline  with  NaOH,  and  a  few  drops 
of  copper  sulphate  solution  added.  A  violet  color  indicates  the  pres- 
ence of  pepton — proving  proteolysis  of  the  original  albumin. 

(Acetic  acid  of  1.04  prepared  by  diluting  400  c.c.  of  cone,  of  Sp.  Gr.  1.75  with  70C 
c.c.  of  water.) 

II.  A-Naphthylamin. — Dissolve  0.1  g.  in  20  c.c.  of  water,  boil,  filter  (if  necessary), 
and  to  clear  filtrate  add  180  c.c.  of  acetic  acid,  Sp.  Gr.  1.04. 

The  solutions  are  kept  separate  and  mixed  in  equal  parts  just  before  use. 

In  carrying  out  the  test,  put  2  c.c.  of  each  reagent  in  a  test  tube  and  add  substance 
to  be  tested.  (In  ordinary  water  analysis  use  100  c.c.)  Cover  tube  with  watch 
glass  and  set  in  warm  water  for  20  minutes.  Observe  presence  or  absence  of  pink 
color  promptly.  Always  run  a  blank  on  the  distilled  water  used  for  rinsing  to  avoid 
errors  due  to  nitrites  in  the  water,  or  in  the  air  of  the  laboratory.. 

^Bitter,  Archiv  f.  Hyg.,  v.  1886. 

*  Hanhin  and  Weshrook^  Ann.  Past.,  vi.,  1892, 


DETERMINING   BIOLOGICAL  ACTIVITIES  OF  BACTERIA        169 


u 


'^^m 


DiASTATic  Enzymes. — The  presence  of  diastatic  ferments  may  be 
determined  by  mixing  broth  cultures  of  the  bacteria  with  thin  starch 
paste.  It  is  necessary  that  both  the  cultures  and  the  starch  paste  be 
absolutely  free  from  sugar.  After  remaining  in  the  incubator  for  five  or 
six  hours,  the  fluid  is  filtered  and  the  filtrate  tested  by  methods  used  for 
determining  the  presence  of  sugars. 

Inverting  Ferments. — Inverting  ferments  are  determined  by  a  pro- 
cedure similar  to  the  above  in  principle.  Dilute  solutions  of  cane  sugar 
are  mixed  with  old  cultures  or 
culture  filtrates  of  the  respective 
bacteria  and  the  mixture  allowed 
to  stand.  It  is  then  filtered, 
and  the  filtrate  tested  for  glucose, 
preferably  by  Fehling's  solution. 

ANIMAL    EXPERIMENTATION 

In  the  study  of  pathogenic 
microorganisms,  animal  experi- 
mentation is  essential  in  many 
instances.  The  virulence  of  any 
given  organism  for  a  definite  ani- 
mal species  and  the  nature  of  the 
lesions  produced  are  character- 
istics often  of  great  value  in 
differentiation.  Isolation,  more- 
over, of  many  bacteria  is  greatly 
facilitated  by  the  inoculation  of 
susceptible  animals  and  recovery 
of  the  pathogenic  organism  from 

the  heart's  blood  or  from  the  lesions  produced  in  various  organs.  That 
investigations  into  the  phenomena  of  immunity  would  be  absolutely 
impossible  without  the  use  of  animal  inoculation  is,  of  course,  self- 
evident,  for  by  this  method  only  can  the  action  of  bacteria  in  relation  to 
living  tissues,  cells,  and  body-fluids  be  observed . 

The  animals  most  commonly  employed  for  such  observations  are 
guinea-pigs,  white  mice,  white  rats,  and  rabbits.  The  method  of 
inoculation  may  be  either  subcutaneous,  intrapleural,  intraperi- 
toneal, intravenous,  or  subdural,  etc.  Tt  must  be  borne  in  mind 
always  that  the  mode  of  inoculation  may  influence  the  course  of  an 


O' 


\^ 


Fig. 


48. — ^Types  op  Gelatin  Liquefac- 
tion BY  Bacteria. 


170 


BIOLOGY  AND  TECHNIQUE 


infection  no  less  than  does  the  virulence  of  the  microorganism  or  the 
size  of  the  dose. 

Inoculations  are  made  with  some  form  of  hypodermic  needle  fitted  to 


Fig.  49. — Intraperitoneal  Inoculation  of   Rabbit, 


Fig.  50. — Intravenous  Inoculation  of  Rabbit. 


a  syringe.  The  most  convenient  syringes  are  the  all-glass  Luer  or  the 
Debove  syringes,  which,  however,  are  expensive. .  Any  form  of  steriliz- 
able  syringe  may  be  used.     In  making  inoculations  the  hair  of  the 


DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERIA        171 

animal  should  be  clipped  and  the  skin  disinfected  with  carbolic  acid 
or  alcohol. 

Subcutaneous  inoculations  are  most  conveniently  made  in  the  abdom- 


FiG.  51. — Intraperitoneal  Inoculation  of  Guinea-pig. 


Fig    52. — Guinea-pig  Cage. 


inal  wall,  where  the  skin  is  thin.      After   chpping  and   sterilizing,  the 
skin  is  raised  between  the  fingers  of  the  left  hand  and  the  needle  plunged 


172 


BIOLOGY  AND  TECHNIQUE 


in  obliquely  so  as  to  avoid  penetrating  the  abdominal  wall  and  entering 
the  peritoneum. 

In  making  intraperitoneal  inoculations,  great  care  must  be  exercised 
not  to  puncture  the  gut.  This  can  be  avoided  by  passing  the  needle 
first  through  the  skin  in  an  oblique  direction,  then  turning  it  into  a  posi- 
tion more  vertical  to  the  abdomen  and  perforating  the  muscles  and  perito- 
neum by  a  very  short  and  carefully  executed  stab. 

Intravenous  inoculations  in  rabbits  are  made  into  the  veins  running 
along  the  outer  margins  of  the  ears.  The  hair  over  the  ear  is  clipped  and 
the  animal  held  for  a  short  time  head  downward  so  that  the  vessels 
of  the  head  may  fill  with  blood.    An  assistant  holds  the  animal  firmly  in 


Fig.  53. — Rabbit  Cage. 


a  horizontal  position,  the  operator  grasps  the  tip  of  the  ears  with  the 
left  hand,  and  carefully  passes  his  needle  into  the  vein  in  the  direction 
as  nearly  as  possible  parallel  to  its  course.     (See  Fig.  50.) 

Mice  are  usually  inoculated  under  the  skin  near  the  base  of  the  tail. 
They  may  be  placed  in  a  jar  over  which  a  cover  of  stiff  wire-gauze  is 
held.  They  are  then  grasped  by  the  tail,  by  which  they  are  drawn  up 
between  the  side  of  the  jar  and  the  edge  of  the  wire  cover,  so  that  the 
lower  end  of  the  back  shall  be  easily  accessible.  The  skin  is  then  wiped 
with  a  piece  of  cotton  dipped  in  carbolic  solution  and  the  needle  is  in- 
serted. Great  care  must  be  exercised  to  avoid  passing  the  needle  too 
close  to  the  vertebral  column.  Mice  are  extremely  delicate,  and  any 
injury  to  the' spine  usually  causes  immediate  death. 


DETERMINING  BIOLOGICAL  ACTIVITIES  OF  BACTERIA        173 

With  proper  care  mice  or  rats  may  be  easily  injected  intravenously 
if  a  sufficiently  fine  needle  is  used.  There  are  four  superficially  placed 
veins  running  along  the  tail,  which  stand  out  prominently  when  rubbed 
with  cotton  moistened  with  xylol.    Into  these  the  injections  are  made. 

When  inoculating  rats  or  guinea-pigs  with  bacillus  pestis  the  KoUe 
vaccination  method  is  used.  The  skin  is  merely  shaved  and  a  loopful 
of  the  culture  vigorously  rubbed  into  the  shaven  area. 

The  various  forms  of  animal  holders  which  have  been  devised  are 
rarely  necessary  in  bacteriological  work  unless  working  unassisted,  im- 
mobilization of  the  animals  being  easily  accomplished  by  the  hands  of  a 
skilled  assistant. 

Autopsies  upon  infected  animals  must  be  carefully  made.  The  ani- 
mals are  tied,  back  down,  upon  pans  fitted  in  the  corners  with  clamps  for 
the  strings.  They  are  then  moistened  either  with  hot  water  or  with  a 
weak  solution  of  carbolic  acid,  so  that  contamination  by  hair  may  be 
avoided.  A  median  cut  is  made,  the  skin  is  carefully  dissected  back, 
and  the  body  cavities  are  opened  with  sterile  instruments.  Cultures 
may  then  be  taken  from  exudates,  blood,  or  organs  under  precautions 
similar  to  those  recommended  below  for  similar  procedures  at  autopsy 
upon  man. 

Inoculated  animals  should  be,  if  possible,  kept  separate  from  healthy 
mimals.  Rabbits  and  guinea-pigs  are  best  kept  in  galvanized  iron-wire 
cages,  which  are  fitted  with  floor-pans  that  can  be  taken  out  and  cleaned 
and  sterilized.  Mice  ma)^  be  kept  in  battery  jars  fitted  with  perforated 
metal  covers.  The  mice  should  be  supplied  with  large  pieces  of  cotton 
upon  batting  since  they  are  delicately  susceptible  to  cold. 


CHAPTER  X 

THE  BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL  FROM 

PATIENTS 

In  making  bacteriological  examinations  of  material  taken  from 
living  patients,  or  at  autopsy,  the  validity  of  result  is  as  fully  dependent 
upon  the  technique  by  which  the  material  is  collected,  as  upon  proper 
manipulation  in  the  later  stages  of  examination. 

Material  taken  at  autopsy  should  be,  if  possible,  directly  transferred 
from  the  cadaver  to  the  proper  culture  media.  If  cultures  are  to  be  taken 
from  the  liver,  spleen,  or  other  organs,  the  surface  of  the  organ  should 
first  be  seared  with  a  hot  scalpel  and  an  incision  made  through  the  cap- 
sule of  the  organ  in  the  seared  area,  with  the  same  instrument.  The 
platinum  needle  can  then  be  plunged  through  this  incision  and  material 
for  cultivatipn  be  taken  with  little  chance  of  surface  contamination. 
When  blood  is  to  be  transferred  from  the  heart,  the  heart  muscle  may  be 
incised  with  a  hot  knife,  or  else  the  needle  of  a  hypodermic  syringe  may 
be  plunged  through  the  previously  seared  heart  muscle  and  the  blood 
aspirated.  The  same  end  can  be  accomplished  by  means  of  a  pointed, 
freshly  prepared  Pasteur  pipette.  In  taking  specimens  of  blood  at  au- 
topsy it  is  safer  to  take  them  from  the  arm  or  leg,  by  allowing  the  blood 
to  flow  into  a  broad,  deep  cut  miade  through  the  sterilized  skin, than  from- 
the  heart,  since  it  has  been  found  that  post-mortem  contamination  of 
the  heart's  blood  takes  place  rapidly,  probably  through  the  large  veins 
from  the  lungs.  Exudates  from  the  pleural  cavities,  the  pericardium, 
or  the  peritoneum  may  be  taken  with  a  sterilized  syringe  or  pipette. 

Materials  collected  at  the  bedside  or  in  the  operating-room  should 
be  transferred  directly  to  the  proper  media  or  else  into  sterile  test  tubes 
and  so  sent  to  the  laborator}^  When  the  material  is  scanty,  it  may  be 
collected  upon  a  sterile  cotton  swab,  which  should  be  immediately  re- 
placed in  the  sterilized  containing  tube  and  sent  to  the  laboratory. 

Syringes,  when  used  for  the  collection  of  exudates  or  blood,  should 
be  of  some  variety  which  is  easily  sterilizable  by  dry  heat,  or  boiling. 
Most  convenient  of  the  forms  in  common  use  are  the  all-glass  "  Luer " 
syringe,  or  the  cheaper  ''Sub-Q"  model.  Instruments  which  can  be 
sterilized  only  by  chemical  disinfectants  should  not  be  used.     When 

174 


EXAMINATION  OF  MATERIAL  FROM  PATIENTS  175 

fluids  are  collected  for  bacteriological  examination,  such  as  spinal  fluid, 
paracentesis  fluid,  or  pleural  exudate,  it  is  convenient  to  have  them  taken 
directly  into  sterilized  centrifuge  tubes,  since  it  is  often  necessary  to 
concentrate  cellular  elements  by  centrifugalization.  By  immediate  col- 
lection in  these  tubes,  the  danger  of  contamination  is  avoided. 

Examination  of  Exudates. — Pus. — Pus  should  first  be  examined 
i^aorphologically  by  some  simple  stain,  such  as  gentian-violet,  and  by 
the  Gram  stain.  It  is  convenient,  also,  to  stain  a  specimen  by  Jenner's 
stain,  in  order  to  show  clearly  the  relation  of  bacteria  to  the  cells. 
Such  morphological  examination  not  only  furnishes  a  guide  to  future 
manipulation,  but  supplies  a  control  for  the  results  obtained  by  cultural 
methods.  Specimens  of  the  pus  are  then  transferred  to  the  proper 
media,  and  pour-plates  made  or  streaks  made  upon  the  surface  of 
previously  prepared  agar  or  serum-agar  plates. 

A  guide  to  the  choice  of  media  is  often  found  in  the  result  of  the 
morphological  examination.  In  most  cases,  it  is  well  also  to  make 
anaerobic  cultures  by  some  simpler  method.     (See  page  148  et  seq.)    ■ 

The  colonies  which  develop  upon  the  plates  should  be  studied  under  the 
microscope,  and  specimens  from  the  colonies  transferred  to  cover-glasses 
and  slides  for  morphological  examination  and  to  the  various  media  for 
further  growth  and  identification.  Animal  inoculation  and  agglutination 
tests  must  often  also  be  resorted  to.  A  knowledge  of  the  source  of  the 
material  may  furnish  considerable  aid  in  making  a  bacteriological  diag- 
nosis, though  great  caution  in  depending  upon  such  aid  is  recommended. 

In  the  examination  of  peritoneal,  pericardial,  or  pleural  exudate  it  is 
often  advantageous  to  use  the  sediment  obtained  by  centrifugalization. 
A  differential  count  of  the  cells  present  may  be  of  aid  in  confirming  the 
bacteriological  findings.  Morphological  examination  and  cultural  exam- 
ination are  made  as  in  the  case  of  pus.  Specimens  should  also  in  these 
cases  be  stained  for  tubercle  bacilli.  Whenever  morphological  exami- 
nations of  such  fluids  are  negative,  no  bacteria  being  found,  and  especially 
when  among  the  cellular  elements  the  lymphocytes  preponderate,  the 
search  for  tubercle  bacilli  should  be  continued  by  means  of  animal  inocu- 
lation. Guinea-pigs  should  be  inoculated  intraperitoneally  from  speci- 
mens of  the  fluid.  The  animals  will  usually  die  within  six  to  eight  weeks, 
but  can  be  killed  and  examined  at  the  end  of  about  six  weeks  if  they 
remain  alive.  The  chances  for  a  positive  result  are  considerably 
increased  if  the  fluid  is  set  away  in  the  ice-chest  until  a  clot  has  formed 
and  the  animals  are  inoculated  with  the  material  from  the  broken-up 
clot  j3 


176  BIOLOGY  AND  TECHNIQUE 

The  routine  examination  of  spinal  fluid  is  best  made  upon  the  sedi- 
ment of  centrifugalized  specimens.  The  microorganisms  with  which  we 
deal  most  frequently  in  this  fluid  are  the  meningococcus,  the  pneumococ- 
cus,  the  streptococcus,  and  the  tubercle  bacillus.  If  morphological  ex- 
amination reveals  bacteria  resembling  the  first  three  of  these  in  appear- 
ance and  staining-reaction,  surface  smears  should  preferably  be  made 
upon  plates  of  serum  agar,  blood  agar,  or  upon  tubes  of  Loeffler's  co- 
agulated blood-serum.  Failure  to  find  organisms  morphologically  does 
not  exclude  their  presence  and  careful  cultivation  should  be  done  in  all 
cases.  When  organisms  are  not  found  by  simple  morphological  examina- 
tion and  the  fluid  and  sediment  are  scanty,  specimens  should  be  stained 
by  the  Ziehl-Neelson  method  for  tubercle  bacilli.  In  such  cases  it  is 
often  of  advantage  to  set  away  the  specimen  until  a  thin  thread-like 
clot  of  fifcrin  has  formed  in  the  bottom  of  the  tube.  In  smears  of  such 
a  clot,  tubercle  bacilli  are  found  with  far  greater  ease  than  they  are  found 
in  centrifugalized  specimens.  If  these  examinations  are  without  result, 
inoculation  of  guinea-pigs  should  be  resorted  to. 

Examination  of  Urine. — Bacteriological  examination  of  the  urine  is 
of  value  only  when  specimens  have  been  taken  with  sterile  catheters, 
and  care  has  been  exercised  in  the  disinfection  of  the  external  genitals. 
Many  of  the  numerous  finds  of  bacillus  coli  in  urine  are  unquestionably 
due  to  defective  methods  of  collecting  material.  Urine  should  be  cen- 
trifugalized and  the  sediment  examined  morphologically  and  pour- 
plates  made  and  surface  smears  made  upon  the  proper  media.  If 
necessary,  animal  inoculation  may  be  done.  In  examining  urine  for 
tubercle  bacilli,  special  care  should  be  taken  in  staining  methods  so 
as  to  differentiate  from  Bacillus  smegmatis. 

Examination  of  Feces. — Human  feces  contain  an  enormous  num- 
ber of  bacteria  of  many  varieties.  Klein,^  by  special  methods,  es- 
timated that  there  were  about  75,000,000  bacteria  in  one  milligram 
of  feces.  It  has  been  a  noticeable  result  of  all  the  investigations  upon 
the  feces,  that  although  enormous  numbers  can  be  counted  in  morpho- 
logical specimens,  only  a  disproportionately  smaller  number  can  be 
cultivated  from  the  same  specimen.  This  is  explicable  upon  the  ground 
that  special  culture  media  are  necessary  for  many  of  the  species  found 
in  intestinal  contents  and  upon  the  consideration  that  many  of  the 
bacteria  which  are  present  in  the  morphological  specimen  are  dead,  show- 
mg  that  there  are  bactericidal  processes  going  on  in  some  parts  of  the 


» Klein,  Ref.  Cent.  f.  Bakt.,  I,  xxx,  1901. 


EXAMINATION  OF    MATERIAL    FROM    PATIENTS  177 

intestinal  tract,  possibly  through  the  agency  of  intestinal  secretions, 
bile,  and  the  action  of  the  products  of  metabolism  of  the  hardier  species 
present.  By  far  the  greater  part  of  the  intestinal  flora  consists  of  mem- 
bers of  the  colon  group,  bacilli  of  the  lactis  aerogenes  group^  Bacillus 
faecalis  alkaligenes,  Bacillus  mesentericus,  and  relatively  smaller  num- 
bers of  streptococci,  staphylococci,  and  Gram-positive  anaerobes.  Many 
other  species,  however,  may  be  present  without  being  necessarily  con- 
sidered of  pathological  significance.  Certain  writers  have  recently  laid 
much  stress  upon  a  preponderance  of  Gram-positive  bacteria  in  speci- 
mens of  feces,  claiming  that  such  preponderance  signifies  some  form 
of  intestinal  disturbance.  Herter  ^  has  recently  advanced  the  opinion 
that  the  presence  of  Bacillus  aerogenes  capsulatus  in  the  intestinal  canal 
is  definitely  associated  with  pernicious  anemia.  The  determination  of 
these  bacilli  in  the  stools  is  made  both  by  morphological  examination 
by  means  of  Gram  stain  and  by  isolation  of  the  bacteria.  Such  isola- 
tion is  easily  done  by  the  method  of  Welch  and  Nuttal.^  A  suspension 
of  small  quantities  of  the  feces  in  salt  solution  is  made  and  1  c.c.  of  the 
filtered  suspension  is  injected  into  the  ear  vein  of  a  rabbit.  After  a  few 
minutes  the  rabbit  is  killed  and  placed  in  the  incubator.  After  five  hours 
of  incubation,  the  rabbit  is  dissected,  and  if  the  Welch  bacillus  has 
been  present  in  the  feces,  small  bubbles  of  gas  will  have  appeared  in 
the  liver  from  which  the  bacilli  may  be  cultivated  in  anaerobic  stab- 
cultures. 

Bacteriological  examination  of  feces  is  most  often  undertaken  for 
the  isolation  of  Bacillus  typhosus.  This  is  accomplished  with  a  great 
deal  of  difficulty  because  of  the  overwhelming  numbers  of  colon  bacilli 
which  easily  outgrow  the  typhoid  germs,  and  because  of  the  similarity 
of  their  colonies  in  most  media.  Many  methods  have  been  devised  for 
this  purpose,  all  of  which  depend  upon  the  use  of  special  media  aimed 
at  the  inhibition  of  colon  and  other  bacilli  and  the  production  of  recog- 
nizable differences  in  the  colonies  of  typhoid  and  colon  bacilli.  Such 
media  are  those  of  Eisner,  Hiss,  Conradi-Drigalski,  Loeffler,  Hesse,  and 
others,  which  are  described  in  the  section  upon  special  media.  (See  page 
133.)  The  methods  of  using  these  media  will  be  found  described  in  the 
chapter  on  Bacillus  typhosus  (p.  399.) 

Cholera  spirilla  may  be  recognized  in  and  isolated  from  the  stools  of 
patients  by  morphological  examination,  and  by  cultivation.  (See 
section  on  Sp.  cholerse.) 

1  Herter,  "  Common  Bacterial  Infections  of  the  Digestive  Tract,"  N.  Y.,  1907. 

2  Welch  and  Nuttal.    See  ref.  p.  469. 


178  BIOLOGY  AND  TECHNIQUE 

For  the  isolation  of  dysentery  bacilli  from  feces,  no  satisfactory 
special  methods  have  as  yet  been  devised.  Here  we  can  depend  only  up- 
on careful  plating  upon  agar  and  gelatin  and  extended  colony  "fishing," 
and  the  study  of  pure  cultures.  The  complete  absence  of  motility  of 
these  bacteria  is  of  much  aid  in  such  identification. 

The  determination  of  tubercle  bacilli  in  stools  is  difficult  and  of 
questionable  significance,  in  that  they  may  be  present  in  people  suffer- 
ing from  pulmonary  tuberculosis  as  a  consequence  of  swallowing  sputum 
or  infected  food,  and  in  that  there  may  be  other  acid-fast  bacilli,  such 
as  the  timothy  bacillus,  present. 

Blood  Cultures. — The  diagnosis  of  septicemia  can  be  positively  made 
during  life  only  by  the  isolation  of  bacteria  from  the  blood.  Such  exam- 
inations are  of  much  value  and  are  usually  successful  if  the  technique 
is  properly  carried  out.  A  large  number  of  methods  are  recommended, 
the  writers  giving,  however,  only  the  one  which  they  have  found 
successful  and  simple  for  general  use. 

The  blood  is  taken  by  preference  from  the  median  basilic  vein  of  the 
arm.  If,  for  some  reason  (both  forearms  having  been  used  for  saline 
infusion),  these  veins  are  unavailable,  blood  may  be  taken  from  the 
internal  saphenous  vein  as  it  turns  over  the  internal  malleolus  of  the 
ankle  joint.  The  skin  over  the  vein  should  be  prepared  before  the 
specimen  is  taken  by  painting  with  iodine,  as  for  a  surgical  operation. 
The  syringe  which  is  used  should  be  of  some  sterilizable  variety  (the 
most  convenient  the  Luer  model),  which  is  easily  manipulated  and 
does  not  draw  with  a  jerky,  irregular  motion.  Its  capacity  should  be 
at  least  10  e.c.  It  may  be  sterilized  by  boiling  for  half  an  hour,  or 
preferably,  when  all-glass  syringes  are  used,  they  may  be  inserted  into 
potato-tubes  and  sterilized  at  high  temperature  in  the  hot-air  chamber. 
Before  drawing  the  blood,  a  linen  bandage  is  wound  tightly  about 
the  upper  arm  of  the  patient  in  order  to  cause  the  veins  to  stand 
out  prominently.  "When  the  veins  are  plainly  in  view,  the  needle  is 
plunged  through  the  skin  into  the  vein  in  a  direction  parallel  to  the 
vessel  and  in  the  direction  of  the  blood-stream.  After  perforation  of 
the  skin,  while  the  needle  is  groping  for  the  vein,  gentle  suction  may 
be  exerted  with  the  piston.  Great  care  should  be  exercised,  however, 
that  the  piston  is  not  allowed  to  slip  back,  and  air  be,  by  accident, 
forced  into  the  vessel.  In  most  cases  no  suction  is  necessary,  the  pres- 
sure of  the  blood  being  sufficient  to  push  up  the  piston.  After  the  blood 
has  been  drawn,  it  should  be  immediately  transferred  to  the  proper 
media.    Epstein  has  recently  recommended  the  mixture  of  the  blood 


EXAMINATION   OF    MATERIAL    FROM    PATIENTS  179 

with  sterile  two  per  cent  ammonium  oxalate  solution  in  test  tubes,  by 
which  means  the  clotting  is  prevented,  and  transfers  can  be  made  more 
leisurely  to  culture  media.  While  this  method  is  convenient  in  cases 
where  blood  must  be  taken  at  some  distance  from  the  laboratory,  it  is 


Fig.  54. — Blood-Culture  Plate  Showing  Streptococcus  Colonies.      Note 
halo  of  hemolysis  about  each  colony. 

preferable,  whenever  possible,  to  make  cultures  from  the  blood  im- 
mediately at  the  bedside. 

The  choice  of  culture  media  for  blood  cultures  should,  to  a  certain 
extent,  be  adapted' to  each  individual  case.  For  routine  work,  it  is  best 
to  employ  glucose-meat-infusion  agar  and  glucose-meat-infusion  broth. 
At  least  six  glucose-agar  tubes  should  be  melted  and  immersed  in  water 
at  45°  C.    Before  the  blood  is  mixed  with  the  medium,  the  agar  should  be 


180  BIOLOGY  AND  TECHNIQUE 

cooled  to  41°  in  order  that  bacteria,  if  present,  may  not  be  injured  by 
the  heat.  The  blood  is  added  to  the  tubes  in  varying  quantities,  ranging 
from  0.25  to  1  c.c.  each,  in  order  that  different  degrees  of  concentration 
may  be  obtained.  Mixing  is  accomplished  by  the  usual  dipping  and 
rotary  motion,  the  formation  of  air-bubbles  being  thus  avoided.  The 
mouth  of  each  test  tube  should  be  passed  through  the  flame  before  pour- 
ing the  contents  into  the  plates.  Three  flasks  of  glucose  broth,  contain- 
ing 100  to  150  c.c.  of  fluid  each,  should  be  inoculated  with  varying 
quantities  of  blood — at  least  one  of  the  flasks  containing  the  blood  in 
high  dilution.  The  most  stringent  care  in  the  withdrawal  and  replace- 
ment of  the  cotton  stoppers  should  be  exercised.'  The  writers  have 
found  it  convenient  to  use,  in  place  of  one  of  these  flasks,  one  containing, 
in  addition  to  the  glucose,  1  gm.  of  powdered  calcium  carbonate. 
This  insures  neutrality,  permitting  pneumococci  or  streptococci,  which 
are  sensitive  to  acid,  to  develop  and  retain  their  vitality. 

In  making  blood  cultures  from  typhoid  patients,  Buxton  and  Cole- 
man ^  have  obtained  excellent  results  by  the  use  of  pure  ox-bile  con- 
taining ten  per  cent  of  glycerin  and  two  per  cent  of  peptone  in  flasks. 
The  writers  have  had  no  difficulty  in  obtaining  typhoid  cultures  by  the 
use  of  slightly  acid  meat-extract  broth  in  flasks  containing  200  or  more 
c.c.  to  which  comparatively  little  blood  has  been  transferred. 

Anaerobic  Blood  Cultures. — These  cultures  may  be  taken  by 
mixing  blood  in  deep  tubes  with  glucose-ascitic  agar,  covering  with 
albolene  and  putting  into  Novy  jars. 

In  estimating  the  results  of  a  blood  culture,  the  exclusion  of  con- 
tamination usually  offers  little  difficulty.  If  the  same  microorganism 
appears  in  several  of  the  plates  and  flasks,  if  colonies  upon  the  plates 
are  well  distributed  within  the  center  and  under  the  surface  of  the 
medium,  and  if  the  microorganisms  themselves  belong  to  species  which 
commonly  cause  septicemia,  such  as  streptococcus  and  pneumococcus, 
it  is  usually  safe  to  assume  that  they  have  emanated  from  the  patient 's 
circulation.  When  colonies  are  present  in  one  plate  or  in  one  flask 
only,  when  they  are  situated  only  near  the  edges  of  a  plate  or  upon 
the  surface  of  the  medium,  and  when  they  belong  to  varieties  which 
are  often  found  saprophytic  upon  skin  or  in  air,  they  must  be  looked 
upon  with  suspicion.  It  is  a  good  rule  to  look  upon  all  staphylococcus 
albus  cultures  skeptically. 

^  Small  Florence  flasks  are  preferable  to  the  Erlenmeyer  flasks  usually  employed. 
2  Buxton  and  Coleman,  Am.  Jour,  of  Med.  Sci.,  1907. 


SECTION  II 

INFECTION    AND    IMMUNITY 


CHAPTER   XI 

FUNDAMENTAL  FACTORS  OF  PATHOGENICITY  AND  INFECTION 

When  microorganisms  gain  entrance  to  the  animal  or  human  body 
and  give  rise  to  disease,  the  process  is  spoken  of  as  infection. 

Bacteria  are  ever  present  in  the  environment  of  animals  and  human 
beings  and  some  find  constant  lodgment  on  various  parts  of  the  body. 
The  mouth,  the  nasal  passages,  the  skin,  the  upper  respiratory  tract,  the 
conjunctivae,  the  ducts  of  the  genital  system,  and  the  intestines  are 
invariably  inhabited  by  numerous  species  of  bacteria,  which,  while  sub- 
ject to  no  absolute  constancy,  conform  to  more  or  less  definite  charac- 
teristics of  species  distribution  for  each  locality.  Thus  the  colon  organ- 
isms are  invariably  present  in  the  normal  bowel,  Doderlein's  bacillus 
in  the  vagina.  Bacillus  xerosis  in  many  normal  conjunctivae,  and  staph}^- 
lococcus,  streptococcus,  various  spirilla,  and  pneumococcus  in  the  mouth. 
In  contact,  therefore,  with  the  bodies  of  animals  and  man,  there  is  a  large 
flora  of  microorganisms,  some  as  constant  parasites,  others  as  transient 
invaders;  some  harmless  saprophytes  and  others  capable  of  becoming 
pathogenic.  It  is  evident,  therefore,  that  the  production  of  an  infection 
must  depend  upon  other  influences  than  the  mere  presence  of  the  micro- 
organisms and  their  contact  with  the  body,  and  that  the  occurrence  of 
the  reaction — for  the  phenomena  of  infection  are  in  truth  reactions  be- 
tween the  germ  and  the  body  defenses — is  governed  by  a  number  of 
important  secondary  factors. 

In  order  to  cause  infection,  it  is  necessary  that  the  bacteria  shall  gain 
entrance  to  the  body  by  a  path  adapted  to  their  own  respective  cultural 
requirements,  and  shall  be  permitted  to  proliferate  after  gaining  a  foot- 
hold. Some  of  the  bacteria  then  cause  disease  by  rapid  multiplication, 
progressively  invading  more  and  more  extensive  areas  of  the  animal 
tissues,  while  others  may  remain  localized  at  the  point  of  invasion  and 

181 


182  INFECTION  AND  IMMUNITY 

exert  their  harmful  action  chiefly  by  local  growth  and  the  elaboration  of 
specific  poisons. 

The  inciting  or  inhibiting  factors  which  permit  or  i3rohibit  an  in- 
fection are  dependent  iri  part  upon  the  nature  of  the  invading  germ  and 
in  part  upon  the  conditions  of  the  defensive  mechanism  of  the  subject 
attacked. 

Bacteria  are  roughly  divided  into  two  classes,  saprophytes  and 
parasites.  The  saprophytes  are  those  bacteria  which  thrive  best  on 
dead  organic  matter  and  fulfill  the  enormously  important  function  in 
nature  of  reducing  by  their  physiological  activities  the  excreta  and 
dead  bodies  of  more  highly  organized  forms  into  those  simple  chemical 
substances  which  may  again  be  utilized  by  the  plants  in  their  con- 
structive processes.  The  saprophytes,  thus,  are  of  extreme  importance 
in  maintaining  the  chemical  balance  between  the  animal  and  plant 
kingdoms.  Parasites,  on  the  other  hand,  find  the  most  favorable 
conditions  for  their  development  upon  the  living  bodies  of  higher  forms. 

While  a  strict  separation  of  the  two  divisions  can  not  be  made,  nu- 
merous species  forming  transitions  between  the  two,  it  may  be  said 
that  the  latter  class  comprises  most  of  the  so-called  pathogenic  or 
disease-producing  bacteria.  Strict  saprophytes  may  cause  disease, 
but  only  in  cases  where  other  factors  have  brought  about  the  death 
of  some  part  of  the  tissues,  and  the  bacteria  invade  the  necrotic 
areas  and  break  down  the  proteids  into  poisonous  chemical  sub- 
stances such  as  ptomains,  or  through  their  own  destruction  give 
rise  to  the  liberation  of  toxic  constituents  of  their  bodies.  It  is 
necessary,  therefore,  that  bacteria,  in  order  to  incite  disease, 
should  belong  strictly  or  facultatively  to  the  class  known  as  para- 
sitic. It  must  not  be  forgotten,  however,  that  the  terms  are  relative, 
and  that  bacteria  ordinarily  saprophytic  may  develop  parasitic 
and  pathogenic  powers  when  the  resisting  forces  of  the  invaded 
subject  ^re  reduced  to  a  minimum  by  chronic  constitutional  disease 
or  other  causes. 

Organisms  that  are  parasitic,  however,  are  not  necessarily  pathoge.ic, 
and  there  are  certain  more  or  less  fundamental  requirements  which 
experience  has  taught  us  must  be  met  by  an  organism  in  order  that  it 
may  be  infectious  (or  pathogenic)  for  any  given  animal;  and  by  infec- 
tiousness is  meant  the  ability  of  an  organism  to  live  and  multiply  in  the 
animal  fluids  and  tissues.  For  instance,  an  organism  which  is  shown 
not  to  grow  at  the  body  temperature  of  warm-blooded  animals  may 
safely  be  assumed  not  to  be  infectious  for  such  animals;  and  experience  is 


FACTORS  OF   PATHOGENICITY  AND   INFECTION  183 

gradually  teaching  us  that  strictly  aerobic  organisms,  those  thriving 
only  in  the  presence  of  free  oxygen  and  not  able  to  obtain  this  gas  in 
available  combination  from  carbohydrates,  can  also  be  safely  excluded 
from  the  infectious  class.  We  have  also  learned  that  anaerobic  organ- 
isms, although  infectious  when  gaining  entrance  to  tissues  not  abun- 
dantly supplied  with  blood,  are  practically  unable  to  multiply  in  the 
blood  stream  and  give  rise  to  generalized  infection. 

The  pathogenic  microorganisms  differ  very  much  among  themselves , 
in  the  degree  of  their  disease-inciting  power.  Such  power  is  known  as 
virulence.  Variations  in  virulence  occur,  not  only  among  different 
species  of  pathogenic  bacteria,  but  may  occur  within  the  same  species. 
Pneumococci,  for  instance,  which  have  been  kept  upon  artificial  media 
or  in  other  unfavorable  environment  for  some  time,  exhibit  less  viru- 
lence than  when  freshly  isolated  from  the  bodies  of  man  or  ani- 
mals. It  is  necessary,  therefore,  in  order  to  produce  infection,- that 
the  particular  bacterium  involved  shall  possess  sufficient  virulence. 

Whether  or  not  infection  occurs  depends  also  upon  the  number  of 
bacteria  which  gain  entrance  to  the  animal  tissues.  A  small  number  of 
bacteria,  even  though  of  proper  species  and  of  sufficient  virulence,  may 
easily  be  overcome  by  the  first  onslaught  of  the  defensive  forces  of  the 
body.  Bacteria,  therefore,  must  be  in  sufficient  number  to  overcome  local 
defenses  and  to  gain  a  definite  foothold  and  carry  on  their  life  processes, 
before  they  can  give  rise  to  an  infection.  The  more  virulent  the  germ, 
other  conditions  being  equal,  the  smaller  the  number  necessary  for  the 
production  of  disease.  The  introduction  of  a  single  individual  of  the 
anthrax  species,  it  is  claimed,  is  often  sufficient  to  cause  fatal  infection; 
while  forms  less  well  adapted  to  the  parasitic  mode  of  life  will  gain  a 
foothold  in  the  animal  body  only  after.the  introduction  of  large  numbers. 

The  Path  of  Infection. — ^The  portal  by  which  bacteria  gain  entrance 
to  the  human  body  is  of  great  importance  in  determining  whether  or  not 
disease  shall  occur.  Typhoid  bacilli  rubbed  into  the  abraded  skin  may 
give  rise  to  no  reaction  of  importance,  while  the  same  microorganism, 
if  swallowed,  may  cause  fatal  infection.  Conversely,  virulent  strepto- 
cocci, when  swallowed,  may  cause  no  harmful  effects,  while  the  same 
bacteria  rubbed  into  the  skin  may  give  rise  to  a  severe  reaction. 

Animals  and  man  are  protected  against  invasion  by  bacteria  in 
various  ways.  Externally  the  body  is  guarded  by  its  coverings  of  skin 
and  mucous  membranes.  When  these  are  healthy  and  undisturbed, 
microorganisms  are  usually  held  at  bay.  While  tHs  is  true  in  a  gen- 
eral  way   bacteria  may  in  occasional  cases  pass  through  uninjured 


184  INFECTION  AND  IMMUNITY 

skin  and  mucosa.  Thus  the  Austrian  Plague  Commission  found  that 
guinea-pigs  could  be  infected  when  plague  bacilli  were  rubbed  into  the 
shaven  skin,  and  there  can  hardly  be  much  doubt  of  the  fact  that 
tubercle  baciUi  may  occasionally  pass  through  the  intestinal  mucosa  into 
the  lymphatics  without  causing  local  lesions. 

Even  after  bacteria  of  a  pathogenic  species,  in  large  numbers  and  of 
adequate  virulence,  have  passed  through  a  locally  undefended  area  in 
the  skin  or  mucosa  of  an  animal  or  a  human  being  by  a  path  most  favor- 
ably adapted  to  them,  it  is  by  no  means  certain  that  an  infection  will 
take  place.  The  bodies  of  animals  and  of  man  have,  as  we  shall  see,  at 
their  disposal  certain  general,  systemic  weapons  of  defense,  both  in  the 
blood  serum  and  the  cellular  elements  of  blood  and  tissues  which,  if 
normally  vigorous  and  active,  will  usually  overcome  a  certain  number 
of  the  invading  bacteria.  If  these  defenses  are  abnormally  depressed, 
or  the  invading  microorganisms  are  disproportionately  virulent  or  plen- 
tiful, infection  takes  place. 

Bacteria,  after  gaining  an  entrance  to  the  body,  may  give  rise  merely 
to  local  inflammation,  necrosis,  and  abscess  formation.  They  may,  on 
the  other  hand,  from  the  local  lesion,  gain  entrance  into  the  lymphatics 
and  blood-vessels  and  be  carried  freely  into  the  circulation,  where,  if  they 
survive,  the  resulting  condition  is  known  as  bacteriemia  or  septicemia. 
Carried  by  the  blood  to  other  parts  of  the  body,  they  may,  under  favor- 
able circumstances,  gain  foothold  in  various  organs  and  give  rise  to 
secondary  foci  of  inflammation,  necrosis,  and  abscess  formation.  Such 
a  condition  is  known  as  pijemia.  The  disease  processes  arising  as  the 
result  of  bacterial  invasion  may  depend  wholly  or  in  part  upon  the 
mechanical  injury  produced  by  the  process  of  inflammation,  the  dis- 
turbance of  function  caused  by  the  presence  of  the  bacteria  in  the  capil- 
laries and  tissue  spaces,  and  the  absorption  of  the  necrotic  products 
resulting  from  the  reaction  between  the  body  cells  and  the  micro- 
organisms. To  a  large  extent,  however,  infectious  diseases  are  char- 
acterized by  the  symptoms  resulting  from  the  absorption  or  diffusion 
of  the  poisons  produced  by  the  bacteria  themselves. 

Bacterial  Poisons. — It  was  plain,  even  to  the  earliest  students  of  this 
subject,  that  mere  mechanical  capillary  obstruction  or  the  absorption 
of  the  products  of  a  local  inflammation  were  insufl^icient  to  explain  the 
profound  systemic  disturbances  which  accompany  many  bacterial  in- 
fections. The  very  nature  of  bacterial  'disease,  therefore,  suggested  the 
presence  of  poisons. 

It  was  in  his  investigations  into  the  nature  of  these  poisons  that 


FACTORS  OF   PATHOGENICITY  AND  INFECTION  185 

Brieger  *  was  led  to  the  discovery  of  th6  ptomains.  These  bodies,  first 
isolated  by  him  from  decomposing  beef,  fish,  and  human  cadavers,  have 
found  more  extended  discussion  in  another  section.  Accurately  classified, 
they  are  not  true  bacterial  poisons  in  the  sense  in  which  the  term  is 
now  employed.  Although  it  is  true  that  they  are  produced  from  pro- 
teid  material  by  bacterial  action,  they  are  cleavage  products  derived 
from  the  culture  medium  upon  the  composition  of  which  their  nature 
intimately  depends.  The  bacterial  poisons  proper,  on  the  other  hand, 
are  specific  products  of  the  bacteria  themselves,  dependent  upon  the 
nature  of  the  medium  only  as  it  favors  or  retards  the  full  development 
of  the  physiological  functions  of  the  microorganisms.  The  poisons,  pro- 
duced to  a  greater  or  lesser  extent  by  all  pathogenic  microorganisms, 
may  be  of  several  kinds.  The  true  toxins,  in  the  specialized  meaning 
which  the  term  has  acquired,  are  soluble,  truly  secretory  products  of 
the  bacterial  cells,  passing  from  them  into  the  culture  medium  during 
their  life.  They  may  be  obtained  free  from  the  bacteria  by  filtration  and 
in  a  purer  state  from  the  filtrates  by  chemical  precipitation  and  a  vari- 
ety of  other  methods.  The  most  important  examples  of  such  poisons 
are  those  elaborated  by  Bacillus  diphtheriae  and  Bacillus  tetani.  If 
cultures  of  these  bacteria  or  of  others  of  this  class  are  grown  in  fluid 
media  for  several  days  and  the  medium  is  then  filtered  through  porce- 
lain candles,  the  filtrate  will  be  found  toxic  often  to  a  high  degree,  while 
the  residue  will  be  either  inactive  or  comparatively  weak.  Moreover, 
if  the  residue  possesses  any  toxicity  at  all,  the  symptoms  evidencing 
this  will  be  different  from  those  produced  by  the  filtrate. 

There  are  other  microorganisms,  however,  notably  the  cholera 
spirillum  and  the  typhoid  bacillus,  in  which  no  such  exotoxins  are 
formed.  If  these  bacteria  are  cultivated  and  separated  from  the  cul- 
ture fluid  by  filtration,  as  above,  the  fluid  filtrate  will  be  toxic  to  only  a 
very  slight  degree,  whereas  the  residue  may  prove  very  poisonous.  In 
these  cases,  we  are  dealing,  evidently,  with  poisons  not  secreted  into  the 
medium  by  the  bacteria,  but  rather  attached  more  or  less  firmly  to  the 
bacterial  body.  Such  poisons,  separable  from  the  bacteria  only  after 
death  by  some  method  of  extraction,  or  by  autolysis,  were  termed  by 
Pfeiffer  endotoxins.  The  greater  number  of  the  pathogenic  bacteria 
seem  to  act  chiefly  by  means  of  poisons  of  this  class.  The  first  to  call 
attention  to  the  existence  of  such  intracellular  poisons  was  Buchner, 
who  formulated  his  conclusions  from  the  results  of  experiments  made 

^  ^Bneger,  "Die  Ptomaine,"  Berlin,  1885  and  1886. 


186  INFECTION  AND  IMMUNITY    ^ 

with  a  number  of  microorganisms,  notably  the  Friedlander  bacillus 
and  Staphylococcus  pyogenes  aureus,  with  dead  cultures  of  which  he 
induced  the  formation  of  sterile  abscesses  in  animals  and  symptoms 
of  toxemia.  The  conception  of  ''endotoxins,"  received  its  clearest 
and  most  definite  expression  in  the  work  of  Pfeiffer^  on  cholera 
poison. 

Some  clarity  of  conception,  based  on  visual  perception,  may  possibly 
be  gained  by  comparing  some  of  the  products  of  pathogenic  bacteria 
with  bacterial  pigments  and  with  insoluble  interstitial  or  intercellular 
substance,  which  may  be  seen  accompanying  bacteria  in  cover-glass 
preparations.  Soluble  toxic  secretions  are  to  be  compared  to  such  pig- 
ments as  the  pyocyanin  of  Bacillus  pyocyaneus,  which  is  so  readily 
soluble  in  culture  media;  endotoxins  proper,  to  pigments  confined  to 
the  bacterial  cell,  or  at  least,  when  secreted,  being  insoluble  in  culture 
media,  such  for  instance  as  the  well-known  red  pigment  of  Bacillus  pro- 
digiosus,  which  may  often  be  seen  free  among  the  bacteria  in  irregular 
red  granules  like  carmine  powder.  That  bodies  such  as  this  latter  might 
be  extruded  from  pathogenic  bacteria  and  not  be  soluble  in  the  usual  cul- 
ture fluids,  is  not  improbable,  and  the  fact  that  more  or  less  insoluble 
interstitial  substances  are  not  infrequent  among  bacteria  is  well  known. 

In  all  bacterial  bodies,  after  removal  of  toxins  and  endotoxins,  a 
certain  proteid  residue  remains  which,  if  injected  into  animals,  may 
give  rise  to  localized  lesions  such  as  abscesses  or  merely  slight  temporary 
inflammations.  The  nature  of  this  residue  has  been  carefully  studied, 
especially  by  Buchner,  who  has  named  it  bacterial  protein  and  he 
believes  the  substance  to  be  approximately  the  same  in  all  bacteria, 
without  specific  toxic  action,  but  with  a  general  ability  to  exert  a  positive 
chemotactic  effect  on  the  white  blood  cells,  thereby  causing  the  forma- 
tion of  pus.  The  nature  of  the  bacterial  proteins  is  by  no  means  clear, 
and  it  is  still  in  doubt  whether  the  separation  of  these  substances  from 
the  endotoxins  can  be  upheld. 

A  number  of  bacteria  may  give  rise  to  both  varieties  of  poisons. 
Thus,  recently,  Kraus  has  claimed  the  discovery  of  a  soluble  toxin  for 
the  cholera  spirillum  and  Doerr  for  the  dysentery  bacillus,  both  of  which 
microorganisms  were  regarded  as  being  purely  of  the  endotoxin-pro- 
ducing  type. 

It  is  plain,  moreover,  that  occasionally  it  may  be  very  difficult  to 
distinguish  between  a  soluble  toxin  and  an  endotoxin.    In  the  filtration 

1  P/eiJfer,  Zeit.  f.  Hyg.,  xl,  1892. 


FACTORS  OF   PATHOGENICITY  AND   INFECTION  187 

experiment  recorded  above,  it  might  well  be  claimed  that  the  toxicity 
of  the  filtrate,  when  not  very  strong,  may  depend  upon  an  extraction 
of  endotoxins  from  the  bodies  of  the  bacteria  by  the  medium.  The 
final  test,  in  such  instances,  lies  in  the  power  of  true  toxins  to  stimu- 
late in  animals  the  production  of  antitoxins;  for,  as  we  shall  see  later, 
the  injection  of  true  soluble  toxins  into  animals  gives  rise  to  antitoxins, 
whereas  the  formation  of  such  neutralizing  bodies  in  the  serum  or  plasma 
does  not,  it  is  claimed,  follow  the  injection  of  endotoxins.  This  distinc- 
tion will  become  clearer  as  we  proceed  in  the  discussion  of  immunity.  It 
must  not  be  forgotten,  however,  that  our  knowledge  of  bacterial  poisons 
is  by  no  means  complete,  and  that  sharp  distinctions  as  those  given  above 
must  be  regarded  to  a  certain  extent  as  tentative. 

In  resistance  to  chemical  action  and  heat,  the  various  poisons  show 
widely  divergent  properties.  As  a  general  rule,  most  true  soluble 
toxins  are  delicately  thermolabile,  they  are  destroyed  by  moderate 
heating,  and  deteriorate  easily  on  standing.  Their  chemical  nature  is 
by  no  means  clear,  but,  on  precipitation  of  toxic  solutions  with  mag- 
nesium sulphate,  these  poisons  come  down  together  with  the  globulins. 
The  nature  of  the  endotoxins  is  still  less  clearly  understood.  Most  of 
them,  while  less  labile  than  the  extracellular  poisons,  are,  nevertheless, 
destroyed  by  exposure  to  70°  C.  On  the  other  hand,  certain  specific  and 
powerful  intracellular  poisons,  like  those  of  the  Gartner  bacillus  of  meat 
poisoning,  may  undergo  exposure  to  even  100°  C.  and  still  retain  their 
toxic  properties.  The  nature  of  each  individual  poison  will  be  discussed 
in  connection  with  its  microorganism. 

The  Mode  of  Action  of  Bacterial  Poisons. — Close  study  of  the  toxic 
products  of  various  microorganisms  has  shown  that  many  of  the  bac- 
terial poisons  possess  a  more  or  less  deKnite  selective  action  upon  special 
tissues  and  organs.  Thus,  certain  soluble  toxins  of  the  tetanus  bacillus 
and  Bacillus  botulinus  attack  specifically  the  nervous  system.  Again, 
certain  poisons  elaborated  by  the  staphylococci,  the  tetanus  bacillus, 
the  streptococci,  and  other  germs,  the  so-called  "hemolysins,"  attack 
primarily  the  red  blood  corpuscles.  Other  poisons  again  act  on  the 
white  blood  corpuscles;  in  short,  the  characteristic  affinity  of  specific 
bacterial  poisons  for  certain  organs  is  a  widely  recognized  fact. 

In  explanation  of  this  behavior,  much  aid  has  been  given  by  the 
researches  of  Meyer, ^  Overton,^  Ehrlich,^  and  others  upon  the  causes  for 

i  Meyer,  Arch.  f.  exper.  Pathol.,  1899,  1901. 
2  Overton,  "Studien  iib.  d.  Narkose,"  Jena,  1901. 
'^hrlich,  "Sauerstoffs-Bediirfniss  des  OrganismuS;"  Berlin,  1885, 


188  IMMUNITY  AND  INFECTION 

the  analogous  selective  behavior  of  various  narcotics  and  alkaloids. 
It  seems  probable,  from  the  researches  of  these  men,  that  the  selective 
action  of  poisons  depends  upon,  the  ability,  chemical  or  physical  or  both, 
of  the  poisons  to  enter  into  combination  with  the  specifically  affected 
cells.  From  the  nature  of  the  combinations  formed,  it  seems  not  unlikely 
that  the  physical  factors,  such  as  solubility  in  the  cell  plasma,  may  also 
play  an  important  part. 

Observations  of  a  more  purely  bacteriological  nature  have  tended 
to  bear  out  these  conclusions.  Wassermann  and  Takaki,^  for  instance, 
have  shown  that  tetanus  toxin,  which  specifically  attacks  the  nervous 
system,  may  be  removed  from  solution  by  the  addition  of  brain  sub- 
stance. Removal  of  the  brain  tissue  by  centrifugation  leaves  the  solu- 
tion free  from  toxin.  In  the  same  way  it  has  been  shown  that  hemo- 
lytic poisons  can  be  removed  from  solutions  by  contact  with  red 
blood  cells,  but  only  when  the  red  blood  cells  of  susceptible  species  are 
employed. 

Similar  observations  have  been  made  in  the  case  of  leukocidin,  a 
bacterial  poison  acting  upon  the  white  blood  cells  specifically.^ 

That  bacterial  poisons  injected  into  susceptible  animals  rapidly 
disappear  from  the  circulation  is  a  fact  which  bears  out  the  view  that 
a  combination  between  affected  tissue  and  toxin  must  take  place. 
Donitz,^  for  instance,  has  shown  that  within  four  to  eight  minutes  after 
the  injections  of  certain  toxins,  considerable  quantities  will  have  dis- 
appeared from  the  circulation.  Conversely,  Metchnikoff  ^  has  ob- 
served that  tetanus  toxin  injected  into  insusceptible  animals  (lizards) 
may  be  detected  in  the  blood  stream  for  as  long  as  two  months  after 
administration. 

1  Wassermann  und  Takaki,  Berl,  klin.  Woch.,  1898. 
^  Sachs,  Hofmeister's  Beitrage,  11,  1902. 
^Donitz,  Deut.  med.  Woch.,  1897. 
*  Metchnikoff,  "L'immimit^  dans  les  malad.  infect," 


CHAPTER  XII 

DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM 

GENERAL   CONSIDERATIONS 

We  have  seen  that  the  mere  entrance  of  a  pathogenic  microorganism 
into  the  human  or  animal  body  through  a  breach  in  the  continuity  of 
the  mechanical  defenses  of  skin  or  mucosa  does  not  necessarily  lead  to 
the  development  of  an  infection.  The  opportunities  for  such  an  invasion 
are  so  numerous,  and  the  contact  of  members  of  the  animal  kingdom  with 
the  germs  of  disease  is  so  constant,  that  if  this  were  the  case,  sooner  or 
later  all  would  succumb.  It  is  plain,  therefore,  that  the  animal  body 
must  possess  mor  subtle  means  of  defense,  by  virtue  of  which  pathogenic 
germs  are,  even  after  their  entrance  into  the  tissues  and  fluids,  dis- 
posed of,  or  at  least  prevented  from  proliferating  and  elaborating  their 
poisons.  The  power  which  enables  the  body  to  accomplish  this  is  spoken 
of  as  resistance.  When  this  resistance,  which  in  some  degree  is  com- 
mon to  all  members  of  the  animal  kingdom,  is  especially  marked,  it  is 
spoken  of  as  "immunity." 

From  this  it  follows  naturally  that  the  terms  resistance  and  immunity, 
as  well  as  their  converse,  susceptibility,  are  relative  and  not  absolute 
terms.  Degrees  of  resistance  exist,  which  are  determined  to  a  certain 
extent  by  individual,  racial,  or  species  peculiarities;  and  persons  or 
animals  are  spoken  of  as  immune  when  they  are  unaffected  by  an  ex- 
posure or  an  inoculation  to  which  the  normal  average  individual  of  the 
same  species  wOuld  ordinarily  succumb.  The  word  does  not  imply, 
however,  that  these  individuals  could  not  be  infected  with  unusually 
virulent  or  large  doses,  or  under  particularly  unfavorable  circumstances. 
Thus,  birds,  while  immune  against  the  ordinary  dangers  of  tetanus  bacilli, 
may  be  killed  by  experimental  inoculations  with  very  large  doses  of 
tetanus  toxin. ^  Similarly,  Pasteur  rendered  naturally  immune  hens 
susceptible  to  anthrax  by  cooling  them  to  a  subnormal  temperature,  and 
Canalis  and  Morpurgo  did  the  same  with  doves  by  subjecting  them  to 
starvation. 

1  Quoted  from  Abel,  KoUe  und  Wassermann,  "Handbuch,"  etc. 
189 


190  INFECTION  AND  IMMUNITY 

Absolute  immunity  is  exceedingly  rare.  The  entire  insusceptibility 
of  cold-blooded  animals  (frogs  and  turtles)  under  normal  conditions  to 
inoculation  with  even  the  largest  doses  of  many  of  the  bacteria  patho- 
genic for  warm-blooded  animals,  and  the  immunity  of  all  the  lower 
animals  against  leprosy,  are  among  the  few  instances  of  absolute  immu- 
nity known.*  Apart  from  such  exceptional  cases,  however,  resistance, 
immunity,  and  susceptibility  must  be  regarded  as  purely  relative  terms. 

The  power  of  resisting  any  specific  infection  may  be  the  natural 
heritage  of  a  race  or  species,  and  is  then  spoken  of  as  natural  immunity. 
It  may,  on  the  other  hand,  be  acquired  either  accidentally  or  artificially 
by  a  member  of  an  ordinarily  susceptible  species,  and  is  then  called 
acquired  immunity. 

Natural  Immunity. — Species  Immunity. — It  is  well  known  that  many 
of  the  infectious  diseases  which  commonly  affect  man,  do  not,  so  far 
as  we  know,  occur  spontaneously  in  animals.  Thus,  infection  with  B. 
typhosus,  the  vibrio  of  cholera,  or  the  meningococcus  occurs  in  ani- 
mals only  after  experimental  inoculation.  Gonorrheal  and  syphilitic 
infection,  furthermore,  not  only  does  not  occur  spontaneously,  but  is 
produced  experimentally  in  animals  with  the  greatest  difficulty — the 
consequent  diseases  being  incomparably  milder  than  those  occurring  in 
man.  Other  diseases,  like  leprosy,  influenza,  and  the  exanthemata,^ 
have  never  been  successfully  transmitted  to  animals. 

Conversely,  there  are  diseases  among  animals  which  do  not  spon- 
taneously attack  man.  Thus,  human  beings  enjoy  immunity  against 
Rinderpest,  and,  to  a  lesser  degree,  against  chicken  cholera. 

Among  animal  species  themselves  great  differences  in  susceptibility 
and  resistance  toward  the  various  infections  exist.  Often-quoted  ex- 
amples of  this  are  the  remarkable  resistance  to  anthrax  of  rats  and  dogs, 
and  the  iminunity  of  the  common  fowl  against  tetanus. 

The  factors  which  determine  these  differences  of  susceptibility  and 
resistance  among  the  various  species  are  not  clearly  understood.  It 
has  been  suggested  that  diet  in  some  instances  may  influence  these  re- 
lations, inasmuch  as  carnivorous  animals  are  often  highly  resistant  to 
glanders,  anthrax,  and  even  tuberculous  infections,  to  which  herbiv- 
orous animals  are  markedly  susceptible.^  It  is  likely,  too,  that  the  great 
differences  between  animals  of  various  species  in  their  metabolism, 
temperature,  etc.,  may  call  for  special  cultural  adaptation  on  the  part 

Lubarsch,  Zeit.  f.  klin.  Mediz.,  xix. 
With  the  possible  exception  of  smallpox. 
3  Hahn,  in  Kolle  und  Wassermann,  vol.  iv. 


DEFENSIVE   FACTORS   OF  THE  ANIMAL  ORGANISM  191 

of  the  bacteria.  The  fact  that  the  bacillus  of  avian  tuberculosis — 
whose  natural  host  has  a  normal  body  temperature  of  40°  C.  and  above 
— will  grow  on  culture  media  at  40  to  50°  C,  whereas  B.  tuberculosis 
of  man  can  not  be  cultivated  at  a  temperature  above  40°  C,  would 
seem  to  lend  some  support  to  this  view.  The  difference  between  warm- 
and  cold-blooded  animals  has  already  been  noted.  The  necessity  for 
cultural  adaptation,  too,  would  seem  to  be  borne  out  by  the  great 
enhancement  observed  in  the  virulence  of  certain  microorganisms  for 
a  given  species  after  repeated  passage  through  individuals  of  this  species. 

Racial  Immunity. — ^Just  as  differences  in  susceptibility  and  im- 
munity exist  among  the  various  animal  species,  so  the  separate  races  or 
varieties  within  the  same  species  may  display  differences  in  their  reac- 
tions toward  pathogenic  germs.  Algerian  sheep,  for  instance,  show 
a  much  higher  resistance  to  anthrax  than  do  our  own  domestic  sheep, 
and  the  various  races  of  mice  differ  in  their  susceptibility  to  anthrax 
and  to  glanders. 

Similar  racial  differences  are  common  among  human  beings.  As  a 
general  rule,  it  may  be  said  that  a  race  among  whom  a  certain  disease 
has  been  endemic  for  many  ages  is  less  susceptible  to  this  disease  than 
are  other  races  among  whom  it  has  been  more  recently  introduced.  The 
appalling  ravages  of  tuberculosis  among  negroes,  American  Indians,  and 
Esquimaux,  bear  striking  witness  to  this  fact.  Conversely,  the  compar- 
ative immunity  of  the  negro  from  yellow  fever,  a  disease  of  the  greatest 
virulence  for  Caucasians,  furnishes .  further  evidence  in  favor  of  this 
opinion.  It  must  not  be  forgotten,  however,  in  judging  of  these  rela- 
tions, that  the  great  differences  in  the  customs  of  personal  and  social 
hygiene  existing  among  the  various  races  may  considerably  affect  the 
transmission  of  disease  and  lead  to  false  conclusions. 

In  so  far  as  the  statement  made  above  is  true,  however,  it  seems  to 
indicate  that  the  endemic  diseases  have  carried  in  their  train  a  certain 
degree  of  inherited  immunity. 

In  other  cases  *— as  in  the  instance  of  the  malaria-immunity  of 
negroes — the  resistance  seems  to  be  acquired  rather  than  inherited,  for, 
as  Hirsch  was  first  to  note,  death  from  this  disease  occurred  frequently 
among  the  children,  while  adult  negroes  were  rarely  attacked. 

DiFFEKENCES  IN  INDIVIDUAL  RESISTANCE. — In  bacteriological  ex- 
perimentation with  smaller  test  animals,  a  direct  ratio  may  often  exist 
between  body  weight  and  dosage  in  determining  the  outcome  of  an 

J  Hahn,  in  Kolle  and  Wassermann,  loc.  cit. 
14 


192  INFECTION  AND  IMMUNITY 

infection,  provided  the  mode  of  inoculation  has  been  the  same  and  the 
virulence  of  the  germ  not  excessive.  It  would  seem,  therefore,  that 
among  these  animals  the  difference  in  resistance  in  the  face  of  an  arti- 
ficial infection  between  individuals  of  the  same  race  is  very  slight. 

In  higher  animals,  however,  especially  in  the  case  of  man,  the  ex- 
istence of  such  apparent  individual  differences  is  a  well-established  fact, 
although  in  judging  of  them  we  must  not  forget  that  the  conditions  of 
infection  are  not  subject  to  the  uniformity  and  control  which  animal 
experimentation  permits.  Of  a  number  of  persons  exposed  to  any 
given  infection  there  are  always  some  who  are  entirely  unaffected  and 
there  are  great  variations  in  the  severity  of  the  disease  in  those  who 
are  attacked.  In  the  absence  of  positive  evidence  in  support  of  the 
direct  inheritance  of  this  individual  immunity,  the  most  reasonable 
explanation  for  such  differences  in  resistance  seems  to  lie  in  attrib- 
uting them  to  individual  variations  in  metabolism  or  body  chem- 
istry. Depressions,  for  instance,  in  the  acidity  of  the  gastric  secretion 
would  predispose  to  certain  infections  of  gastro-intestinal  origin.  Ana- 
tomical differences,  too,  may  possibly  influence  resistance.  Thus, 
Birch-Hirschfeld  believed  that  certain  anomalous  arrangements  of  the 
bronchial  tubes  predisposed  to  tuberculosis. 

Instances  of  transient  susceptibility  induced  by  physical  or  mental 
overwork,  starvation,  etc.,  should  hardly  be  classified  under  this  head- 
ing, since  the  conditions  in  such  cases  correspond  simply  to  experi- 
mental depression  of  natural  species  or  race  resistance. 

Acquired  Immunity. — It  is  a  matter  of  common  experience  that  many 
of  the  infectious  diseases  occur  but  once  in  the  same  individual.  This 
is  notably  the  case  with  typhoid  fever,  yellow  fever,  and  most  of  the 
exanthemata,  and  is  too  general  an  observation  to  require  extensive 
illustration.  A  single  attack  of  any  of  the  diseases  of  this  class  alters  in 
some  way  the  resistance  of  the  individual  so  that  further  exposure  to 
the  infective  agent  is  usually  without  menace,  either  for  a  limited  period 
after  the  attack,  or  for  life.  Resistance  acquired  in  this  way  is  often 
spoken  of  as  acquired  immunity. 

The  protection  conferred  by  certain  diseases  against  further  attack 
was  recognized  many  centuries  ago,  and  there  are  records  which  show 
that  attempts  were  made  in  ancient  China  and  India  to  inoculate  healthy 
individuals  with  pus  from  small-pox  pustules  in  the  hope  of  producing 
by  this  process  a.  mild  forn>of  the  disease  and  its  consequent  immunity. 

Pasteur,  before  all  others,  thought  philosophically  about  the  phenom- 
ena of  acquired  immunity,  and,  with  adequate  knowledge,  realized  the 


DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM   •        193 

possibility  of  artifically  bestowing  immunity  without  inflicting  the 
dangers  of  the  fully  potent  infective  agent.  The  first  observation  which, 
made  by  him  purely  accidentally,  inspired  the  hope  of  the  achievement 
of  such  a  result,  occurred  during  his  experiments  with  chicken  cholera. 
The  failure  of  animals  to  die  after  inoculation  with  an  old  culture  of  the 
bacilli  of  chicken  cholera,  fully  potent  but  a  few  weeks  previously, 
pointed  to  the  attenuation  of  these  bacilli  by  their  prolonged  cultivation 
without  transplantation.  With  this  observation  as  a  point  of  departure 
he  carried  out  a  series  of  investigations  with  the  purpose  of  discovering 
a  method  of  so  weakening  or  attenuating  various  incitants  of  disease 
that  they  could  be  introduced  into  susceptible  individuals  without  en- 
dangering life  and  yet  without  losing  their  property  of  conferring  pro- 
tection. The  brilliant  results  achieved  by  Jenner,  many  years  before, 
in  protecting  against  smallpox  by  inoculating  with  the  entirely  innocu- 
ous products  of  the  pustules  of  cowpox  furnished  an  analogy  which 
gave  much  encouraging  support  to  this  prospect. 

The  experimental  work  which  Pasteur  carried  out  to  solve  this  prob- 
lem not  only  reaped  a  rich  harvest  of  facts,  but  gave  to  science  the  first 
and  brilliant  examples  of  the  application  of  exact  laboratory  methods  to 
problems  of  immunity. 

ACTIVE   IMMUNITY  ^ 

Active  Artificial  Immunity. — The  process  of  conferring  protection 
by  treatment  with  either  an  attenuated  form  or  a  sublethal  quantity 
of  the  infectious  agent  of  a  disease,  or  its  products,  is  spoken  of  as  "  active 
immunization." 

Whatever  the  method  employed*  the  immunized  individuals  gain 
their  power  of  resistance  by  the  unaided  reactions  of  their  own  tissues. 
They  themselves  take  an  active  physiological  part  in  the  acquisition  of 
this  new  property  of  immunity.  For  this  reason,  Ehrlich  has  aptly 
termed  these  processes  "active  immunization." 

There  are  various  methods  by  which  this  can  be  accomplished,  all 
of  which  were,  in  actual  application  or  in  principle,  discovered  by 
Pasteur  and  his  associates,  and  can  be  best  reviewed  by  a  study  of  their 
work. 

Active  Immunization  with  Attenuated  Cultures. — In  the  course 
of  his  experiments  upon  chicken  cholera,  as  mentioned  above,  Pasteur  * 

1  Pasteur,  Compt.  rend,  de  I'acad.  des  sci.,  1880,  t.  xa. 


194  INFECTION  AND   IMMUNITY 

accidentally  discovered  that  the  virulence  of  the  bacilli  of  this  disease 
was  greatly  reduced  by  prolonged  cultivation  upon  artificial  media. 
This  was  especially  noticeable  in  broth  cultures  which  had  been  stored 
for  long  periods  without  transplantation.  By  repeated  injections  of  such 
cultures  into  fowl,  he  succeeded  in  rendering  the  animals  immune  against 
subsequent  inoculations  with  lethal  doses  of  fully  virulent  strains. 

During  the  same  year,  1880,  in  which  Pasteur  published  his  observa- 
tions on  chicken  cholera,  Toussaint  ^  succeeded  in  immunizing  sheep 
against  anthrax  by  inoculating  them  with  blood  from  infected  animals, 
defibrinated  and  heated  to  55°  *C.  for  ten  minutes.  Toussaint  wrongly 
believed,  however,  that  the  blood  which  had  been  used  in  his  immuniza- 
tions was  free  from  living  bacteria.  In  repeating  this  work  Pasteur 
showed  that  the  protection  in  Toussaint's  cases  was  conferred  by  living 
bacteria,  the  virulence  of  which  had  been  reduced  by  their  subjection  to 
heat. 

In  following  out  the  suggestions  offered  by  these  experiments, 
Pasteur^  discovered  that  he  could  reduce  the  virulence  of  anthrax 
bacilli  much  more  reliably  than  by  Toussaint's  method,  by  cultivating 
the  organisms  at  increased  temperatures  (42°  to  43°  C.) .  By  this  process 
of  attenuation  he  was  able  to  produce  '^  vaccines  "  of  roughly  measurable 
strength,  with  which  he  succeeded  in  immunizing  sheep  and  cattle. 
A  successful  demonstration  of  his  discovery  was  made  by  him  at  Pouilly- 
le-Fort,  soon  after,  upon  a  large  number  of  animals  and  before  a  commis- 
sion of  professional  men. 

It  is  a  fact  well  known  to  bacteriologists  that  certain  of  the  pathogenic 
microorganisms,  when  passed  through  several  individuals  of  the  same 
animal  species,  become  gradually  more  virulent  for  this  species.  In  his 
studies  on  the  bacillus  of  hog  cholera,  Pasteur  observed  that  when  this 
microorganism  was  passed  through  the  bodies  of  several  rabbits  it  gained 
in  virulence  for  rabbits,  but  became  less  potent  against  hogs.  He  suc- 
ceeded, subsequently,  in  protecting  hogs  against  fully  virulent  cultures 
by  treating  them  with  strains  which  had  been  attenuated  by  their 
passage  through  rabbits. 

A  further  principle  of  attenuation  for  purposes  of  immunization  was, 
at  about  this  time,  contributed  by  Chamberland  and  Roux,*  who  re- 


1  Toussaint,  Compt.  rend,  de  Tacad.  des  sci.,  1880,  t.  xci. 

2  Pasteur,  Ch/imberland  et  Roux,  Compt.  rend,  de  Tacad.  des  sci.,  1881,  t.  xeii. 

3  Pasteur,  Compt.  rend,  de  I'acad.  des  sci.,  1882,  t.  xcv.   . 

*  Chamberland  et  Roux,  Compt.  rend,  de  Tacad.  des  sci.,  1882,  t.  xcvi. 


DEFENSIVE  FACTORS   OF  THE  ANIMAL  ORGANISM  195 

duced  the  virulence  of  anthrax  cultures  by  growing  them  in  the  presence 
of  weak  antiseptics  (carbolic  acid  1  :  600,  potassium  bichromate  1  :  5,000, 
or  sulphuric  acid  1  :  200) .  Cultivated  under  such  conditions  the  bacilli 
lost  their  ability  to  form  spores  and  became  entirely  avirulent  for  sheep 
within  ten  days.  A  similar  result  was  later  obtained  by  Behring  ^  when 
attenuating  B.  diphtherise  cultures  by  the  addition  of  terchlorid  of 
iodin. 

Active  Immunization  wim  Sublethal  Doses  of  Fully  Virulent 
Bacteria. — ^The  use  of  fully  virulent  microorganisms  in  minute 
quantities  for  purposes  of  immunization  was  first  suggested  by  Chau- 
veau,2  and  is  naturally  inappHcable  to  extremely  virulent  organisms 
like  B.  anthracis.  The  principle,  however,  is  perfectly  valid,  and  has 
been  experimentally  applied  by  many  observers,  notably  by  Ferran  ^ 
in  the  case  of  cholera.  A  similar  method  proved  of  practical  value  in 
the  hands  of  Theobald  Smith  and  Kilborne  *  in  prophylaxis  against  the 
protozoan  disease,  Texas  fever. 

Active  Immunization  with  Dead  Bacteria. — Suggested  by  Chau- 
veau,  the  method  of  active  immunization  with  gradually  increasing  doses 
of  dead  microorganisms  has  been  successfully  employed  by  various  ob- 
servers, chief  among  whom  are  Pfeiffer,  Brieger,  Wright,  and  Wasser- 
mann.  The  method  is  especially  useful  against  that  class  of  bacteria 
in  which  the  cell  bodies  (endotoxins)  have  been  found  to  be  incomparably 
more  poisonous  than  their  extracellular  products  (toxins).  From  a 
practical  point  of  view,  the  method  is  of  the  greatest  importance  in 
routine  laboratory  immunization  against  B.  typhosus.  Vibrio  cholerse 
asiaticse,  B.  pestis,  and  a  number  of  other  bacteria.  In  the  therapy 
of  human  disease,  this  method  has  recently  come  into  great  prominence, 
chiefly  through  the  work  of  Wright,  whose  investigations  will  be  more 
fully  discussed  in  a  subsequent  section. 

Active  Immunization  with  Bacterial  Products. — Many  bacteria 
when  grown  in  fluid  media  produce  extracellular,  soluble  poisons  which 
remain  in  the  medium  after  the  microorganisms  have  been  removed  by 
filtration  or  centrifugalization.  Since  the  diseases  caused  by  such 
microorganisms  are,  to  a  large  extent,  due  to  the  soluble  poisons  excreted 
by  them,  animals  can  be  actively  immunized  against  this  class  of  bac- 

1  Behring,  Zeit.  f.  Hyg.,  xii,  1892. 

2  Chauveau,  Compt.  rend,  de  I'acad.  des  sci.,  1881,  t.  xcii. 
8  Ferran,  Compt.  rend,  de  I'acad.  des  sci.,  1895,  t.  ci. 

*  Th.  Smith  and  Kilborne,  U.  S.  Dept.  of  Agri».  Bureau  of  Ani.  Indust.,  Wash., 
1893. 


196  INFECTION  AND   IMMUNITY 

teria  by  the  inoculation  of  gradually  increasing  doses  of  the  specific 
poison  or  toxin.  This  method  is  naturally  most  successful  against  those 
microorganisms  which  possess  the  power  of  toxin  formation  to  a  highly 
developed  degree.  Most  important  among  these  are  B.  diphtherias 
and  B.  tetani.  The  first  successful  application  of  this  principle  of  active 
immunization,  however,  was  made  by  Salmon  and  Smith^  in  the  case  of 
hog  cholera. 

PASSIVE    IMMUNITY 

In  Pasteur's  basic  experiments,  as  in  those  of  the  other  scientists  who 
followed  in  his  footsteps,  the  methods  of  immunization  were  based  upon 
the  development  of  a  high  resistance  in  the  treated  subject  by  virtue  of 
its  own  physiological  activities.  This  process  we  have  spoken  of  as? 
"  active  immunization  "  and  it  is  self-evident  that  a  method  of  this  kind 
can.  in  the  treatment  of  disease,  be  employed  prophylactically  onl} 
against  possible  infection,  or  in  localized  acute  infections,  or  at  thp 
beginning  of  a  long  period  of  incubation  before  actual  symptoms  have 
appeared,  as  in  rabies  or  in  chronic  conditions  in  which  the  infection  if 
not  of  a  severe  or  acute  nature. 

A  new  and  therapeutically  more  hopeful  direction  was  given  to  the 
study  of  immunity  when,  in  1890  and  1892,  v.  Behring  and  his  collabora- 
tors discovered  that  the  sera  of  animals  immunized  against  the  toxins 
of  tetanus  ^  and  of  diphtheria  ^  bacilli  would  protect  normal  animals 
against  the  harmful  action  of  these  poisons.  The  animals  thus  pro- 
tected obviously  had  taken  no  active  part  in  their  own  defense,  but 
were  protected  from  the  action  of  the  poison  by  the  substances  trans- 
ferred to  them  in  the  sera  of  the  actively  immunized  animals.  Such 
immunity  or  protection,  therefore,  is  a  purely  passive  phenomenon 
so  far  as  the  treated  animal  is  concerned,  and  the  process  is  for  this 
reason  spoken  of  as  "passive  immunization." 

Passive  immunization  of  this  description  is  practically  applicable 
chiefly  against  diseases  caused  by  bacteria  which  produce  powerful 
toxins,  and  the  sera  of  animals  actively  immunized  against  such  toxins 
are  called  antitoxic  sera.  In  the  treatment  of  the  two  diseases  men- 
tioned above,  diphtheria  and  tetanus,  the  respective  antitoxic  sera  have 


» Salmon  and  Smith,  Rep.  of  Com.  of  Agri.,  Wash.,  1885  and  1886. 
2  V.  Behring  and  Kitasato,  Deut.  med.  Woch.,  49,  1890. 
»v.  Behring  and  Wernicke,  Zeit.  f.  Hyg.,  1892. 


DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM  197 

reached  broad  and  beneficial  therapeutic  application,  and  innumerable 
lives  have  been  saved  by  their  use. 

Passive  immunization  against  microorganisms  not  characterized  by 
marked  toxin  formation  was  attempted,  even  before  Behring's  dis- 
covery, by  Richet  and  Hericourt,^  experimenting  with  cocci,  and  by 
Babes,^  in  the  case  of  rabies;  and  the  underlying  thought  had  been  the 
basis  of  Toussaint's  work  upon  anthrax.  Microorganisms,  however, 
which  exert  their  harmful  action  rather  by  the  contents  of  the  bacterial 
cells  than  by  secreted,  soluble  toxins,  do  not,  so  far  as  is  known,  pro- 
duce antitoxins  in  the  sera  of  immunized  animals.  The  substances 
which  they  call  forth  in  the  process  are  directed  against  the  invading 
organisms  themselves  in  that  they  possess  the  power  of  destroying  or 
of  causing  dissolution  of  the  specific  germs  used  in  their  production. 

Such  antibacterial  sera  are  extensively  used  in  the  laboratory  in  the 
passive  immunization  of  animals  against  a  large  number  of  germs,  and 
are  fairly  effectual  w^hen  used  before,  at  the  same  time  with,  or  soon  after, 
infection.  Their  therapeutic  use  in  human  disease,  however,  has,  up 
to  the  present  time,  been  disappointing  and  their  prophylactic  and  cura- 
tive action  has  been  almost  invariably  ineffect\ial  or  feeble  at  best,  ex- 
cept when  the  antibacterial  sera  could  be  brought  in  direct  contact 
with  the  germs,  in  closed  cavities  or  localized  lesions.  Thus,  in  epidemic 
meningitis,  such  sera  have  proved  extremely  useful  in  the  hands  of 
Flexner,  when  injected  directly  into  the  spinal  canal. 

ANTIBODIES  AND  THE  SUBSTANCES  GIVING  RISE  TO  THEM 

In  the  foregoing  sections  we  have  seen  that  the  process  of  active 
immunization  so  changes  the  animal  body  that  it  becomes  highly 
resistant  against  an  infection  to  which  it  had  formerly  in  many  in- 
stances been  delicately  susceptible.  In  the  absence  of  visible  anatomical 
or  histological  changes  accompanying  the  acquisition  of  this  new  power, 
investigators,  in  order  to  account  for  it,  were  led  to  examine  the  physio- 
logical properties  of  the  body  cells  and  fluids  of  immunized  subjects. 
While  it  was  reasonable  to  suppose  that  all  the  cells  and  tissues  were 
affected  by,  or  might  have  taken  part  in,  a  physiological  change  so 
profoundly  influencing  the  .individual,  the  blood,  because  of  its  unques- 
tionably close  relation  to  inflammatory  reactions,  and  because  of  the 

f  Richet  et  Hericourt,  Compt.  rend,  de  Tacad.  des  sci.,  1888. 
«  Babes  et  Lepp,  Ann.  de  Tinst.  Pasteur,  1889. 


198  INFECTION  AND  IMMUNITY 

ease  with  which  it  could  be  obtained  and  studied,  claimed  the  first 
and  closest  attention.  The  bactericidal  properties  of  normal  blood 
serum  noted  in  1886  by  Nuttall/  v.  Fodor/  and  Fliigge,  moreover,  aided 
in  pointing  to  this  tissue  as  primarily  the  seat  of  the  immunizing 
agents.  It  is  an  interesting  historical  fact,  that,  long  before  this  time, 
the  EngHsh  physician  Hunter  had  noted  that  blood  did  not  decompose 
so  rapidly  as  other  animal  tissues. 

The  study  of  the  blood  serum  of  immunized  animals  as  to  simple 
changes  in  chemical  composition  or  physical  properties  has  shed  little 
light  upon  the  subject.  Beljaeff  ^  in  a  recent  investigation  found  little 
or  no  alteration  from  the  normal  in  the  blood  sera  of  immunized  animals 
as  to  index  of  refraction,  specific  gravity,  and  alkaHnity.  Joachim  ^  and 
Moll  agree  in  stating  that  immune  blood  serum  is  comparatively  richer 
in  globulin  than  normal  serum.  Similar  observations  had  been  made 
by  Hiss  and  Atkinson  ^  and  others.  Important  and  significant  as  these 
purely  chemical  observations  are,  they  have  helped  little  in  explaining 
the  nature  of  the  processes  going  on  in  immune  sera.  The  first  actual 
light  was  thrown  upon  the  mysterious  phenomena  of  immunity  by 
the  investigations  of  Nuttall,^  v.  Fodor,  Buchner,  and  others,  who  not 
only  demonstrated  the  power  of  normal  blood  serum  to  destroy  bacteria, 
but  also  showed  that  this  property  of  blood  serum  became  diminished 
with  age  and  was  destroyed  completely  by  heating  to  56°  C.  The 
thermolabile  substance  of  the  blood  serum  possessing  this  power  was 
called  by  Buchner,^  alexin. 

Soon  after  this  work,  Behring,  in  collaboration  with  Kitasato  ^  and 
Wernicke,*  in  1890  and  1892,  made  further  important  advances  in  the 
elucidation  of  the  immunizing  processes  by  showing  that  the  blood  sera 
of  animals  actively  immunized  against  the  toxins  of  diphtheria  and  tet- 
anus would  protect  normal  animals  against  the  poisons  of  these  diseases. 
He  believed,  at  the  time  of  discovery,  that  such  sera  contained  substances 
which  had  the  power  of  destroying  the  specific  toxins  which  had  been 


1  Nuttall,  Zeit.  f.  Hyg.,  i,  1886. 

2  V.  Fodor,  Deut.  med.  Woch.,  1886. 

3  Beljaeff,  Cent.  f.  Bakt.,  xxxiii. 

*  Joachim,  Pflugers  Archiv,  xciii. 

6  Hiss  and  Atkinson,  Jour.  Exper,  Med.,  v,  1900. 

6  Nuttall,  Zeit.  f.  Hyg.,  1886. 

7  Buchner,  Cent.  f.  Bakt.,  i,  1889. 

8  Behring  und  Kitasato,  Deut.  med.  Woch.,  1890,  No.  49. 

•  Behring  und  Wernicke,  Zeit.  f.  Hyg..  1892 


DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM  199 

used  in  the  immunization.  He  called  these  bodies  antitoxins.  While 
Behring's  first  conception  of  actual  toxin  destruction  soon  proved  to 
be  erroneous,  his  discovery  of  the  presence  in  immune  sera  of  bodies 
specifically  antagonistic  to  toxins  was  soon  confirmed  and  extended, 
and  stands  to-day  as  an  established  fact. 

Ehrlich,^  soon  after  Behring's  announcement,  showed  that  specific 
antitoxins  could  also  be  produced  against  the  poisons  of  some  of  the 
higher  plants  (antiricin,  antikrotin,  antirobin) ,  and  Calmette  ^  produced 
similar  antitoxins  against  snake  poison  (antivenin) .  Stimulated  by  these 
researches,  other  observers  have,  since  then,  added  extensively  to  the 
list  of  poisons  against  which  antitoxins  can  be  produced.  Kempner  ^ 
has  produced  antitoxin  against  the  poison  of  Bacillus  botulinus,  and 
Wassermann,*  against  that  of  Bacillus  pyocyaneus.  Antitoxin  has  been 
produced  by  Calmette  ^  against  the  poison  of  the  scorpion,  and  by  Sachs  ^ 
against  that  of  the  spider.  Thus  a  large  number  of  poisons  of  animal, 
plant,  or  bacterial  origin  have  been  found  capable  of  causing  the  pro- 
duction of  specific  antibodies  in  the  sera  of  animals  into  which  they  are 
injected. 

The  formation  of  antitoxins  directed  against  soluble  poisons,  how- 
ever, did  not  explain  the  immunity  acquired  by  animals  against  bacteria 
like  Bacillus  anthracis,  the  cholera  vibrio,  and  others  which,  unlike  diph- 
theria and  tetanus,  produced  little  or  no  soluble  toxin.  It  was  evident 
that  the  antitoxic  property  of  immune  blood  serum  was  by  no  means 
the  sole  expression  of  its  protective  powers.  Much  light  was  shed  upon 
this  phase  of  the  subject  by  the  discoveries  of  Pfeiffer  in  1894,  who 
worked  along  the  lines  suggested  by  the  investigations  of  Nuttall  and 
Buchner.  Pfeiffer  ^  showed  that  when  cholera  spirilla  were  injected  into 
the  peritoneal  cavity  of  cholera-immune  guinea-pigs,  the  microorganisms 
rapidly  swelled  up,  became  granular,  and  often  underwent  complete 
solution.  The  same  phenomenon  could  be  observed  when  the  bacteria 
were  injected  into  a  normal  animal  together  with  a  sufficient  quantity 
of  cholera-immune  ^  serum. 


*  Ehrlich,  Deut.  med.  Woch.,  1891. 

^  Calmette,  Compt.  rend,  de  la  soc.  de  biol.,  1894. 
^Kempner,  Zeit.  f.  Hyg.,  1897. 

*  Wassermann,  Zeit.  f.  Hyg.,  xxii. 

^  Calmette,  Ann.  de  Tinst.  Pasteur,  1898. 

•Sachs,  Hofm.  Beit.,  1902. 

» Pfeiffer,  Zeit.  f.  Hyg.,  xviii,  1894. 

*  Pfeiffer  und  Isaeff,  ibid. 


200  INFECTION  AND  IMMUNITY 

This  process  lie  observed  microscopically  by  abstracting,  from  time 
to  time,  a  small  quantity  of  the  peritoneal  exudate  and  studying  it  in 
hanging-drop  preparations.  The  reaction  was  specific  in  that  the  de- 
structive process  took  place  to  any  marked  extent  only  in  the  case  of  the 
bacteria  employed  in  the  immunization. 

Metchnikoff ,^  Bordet,  and  others  not  only  confirmed  Pfeiffer's  obser- 
vation, but  were  able  to  show  that  the  lytic  process  would  take  place 
in  vitro,  as  well  as  in  the  animal  body.  The  existence  of  a  specific 
destructive  process  in  immune  serum  was  thus  established  for  the  vibrio 
of  cholera  and  soon  extended  to  other  microorganisms.  The  constitu- 
ents of  the  blood  serum  which  gave  rise  to  this  destructive  phenomenon 
were  spoken  of  as  hacteriolysins. 

Following  closely  upon  the  heels  of  Pfeiifer's  observation  came  the' 
discovery  of  another  specific  property  of  immune  serum  by  Gruber  and 
Durham.^  These  workers  noticed  that  certain  bacteria,  when  brought 
into  contact  with  the  serum  of  an  animal  immunized  against  them, 
were  clumped  together,  deprived  of  motility,  and  firmly  agglutinated. 
They  spoke  of  the  phenomenon  as  agglutination  and  of  the  substances 
in  the  serum  giving  rise  to  it  as  agglutinins. 

The  list  of  antibodies  was  again  enlarged  by  Kraus,^  who  in  1897 
showed  that  precipitates  were  formed  when  filtrates  of  cultures  of 
cholera,  typhoid,  and  plague  bacilli  were  mixed  with  their  specific 
immune  sera.  He  called  the  substances  which  bestowed  this  property 
upon  the  sera  precipitins. 

The  treatment  of  the  animal  body,  therefore,  with  bacteria  or  their 
products  gives  rise  to  a  variety  of  reactions  which  result  in  the  presence 
of  the  ".  antibodies  "  described  above.  Extensive  investigation  has  shown, 
however,  that  the  power  of  stimulating  antibody  production  is  a  phe- 
nomenon not  limited  to  bacteria  and  their  products  alone.  Antitoxins, 
we  have  already  seen,  may  be  produced  with  a  variety  of  poisons  of 
plant  and  animal  origin.  Lysins,  agglutinins,  and  precipitins,  likewise, 
may  be  produced  by  the  use  of  a  large  number  of  different  substances. 
Chief  among  these,  because  of  the  great  aid  they  have  given  to  the  theo- 
retical investigation  of  the  phenomena  of  immunity,  are  the  red  blood 
cells.   Bordet  ^  and,  independently  of  him,  Belfanti  and  Carbone  ^  showed 

^  Metchnikoff^  Ann.  de  Tinst.  Pasteur,  1895. 

2  Gruber  und  Durham,  Miinch.  med.  Woch.,  1896. 

^  Kraus,  R.,  Wien.  klin.  Woch.,  32,  1897. 

*  Bordet,  Ann.  de  Tinst.  Pasteur,  1898. 

6  Belfanti  et  Carhone,  Giornale  della  R.  Acad,  di  Torino,  July,  1^98. 


DEFENSIVE  FACTORS  OF  THE  ANIMAL  ORGANISM  201 

in  1898  that  the  serum  of  animals  repeatedly  injected  with  the  defibri- 
nated  blood  of  another  species  exhibited  the  specific  power  of  dissolving 
the  red  blood  corpuscles  of  this  species.  This  was  the  first  demonstration 
of  "  hemolysis  " — a  phenomenon  which,  because  of  the  ease  with  which 
it  can  be  observed  in  vitro,  has  much  facilitated  investigation.  c^n  Oi 

The  knowledge  that  specific  "endotoxins''  or  cell-destroying  anti-  irt>-^>^ 
bodies  could  be  produced  by  injection  of  red  blood  cells  naturally  sug-  A'UpuJ; 
gested  the  possibility  of  analogous  reactions  for  other  tissue  cells.  It"^^. 
was  not  long,  therefore,  before  Metchnikoff  *  and,  independently  of 
him,  Landsteiner  ^  succeeded,  by  repeated  injections  of  spermatozoa,  in 
producing  a  serum  which  would  seriously  injure  these  specialized  cells. 
Von  Dungem  ^  obtained  similar  results  with  the  ciliated  epithelium  of 
the  trachea.  Since  then  a  host  of  cytotoxins  have  been  produced  with 
the  cells  of  various  organs  and  tissues.  Thus,  Neisser  and  Wechsberg  * 
produced  leucotoxin  (leucocytes);  Delezenne,^  neurotoxin  and  hepa- 
totoxin;  Surmont,^  pancreas  cytotoxin;  and  Bogart  and  Bernard,^  su- 
prarenal cytotoxin. 

One  of  the  most  interesting  of  the  cytotoxins,  moreover,  is  nephro- 
toxin — ^produced  by  the  treatment  of  animals  with  injections  of  emul- 
sions of  kidney  tissue. 

In  all  cases  it  was  supposed  by  those  first  working  with  these  bodies, 
that  the  injection  of  the  sera  of  animals  previously  treated  with  any 
particular  tissue  substance  would  produce  specific  injury  upon  the  or- 
gans homologous  to  the  ones  used  in  immunization.  It  need  hardly  be 
pointed  out  how  very  important  such  phenomena  would  be  in  throwing 
light  upon  the  degenerative  pathological  lesions  occurring  in  disease. 
As  a  matter  of  fact,  however,  sera  so  produced  have  been  shown  to  be 
specific  for  certain  organs  in  a  limited  sense  only.  The  question  of 
specific  cytotoxins  has  been  of  especial  importance  in  the  case  of 
nephritis,  where  Ascoli  and  Figari  ^  and  others  have  suggested  an 
autonephrotoxin  as  the  basis  of  the  pathology  of  this  disease.  In 
the  hands   of    Pearce    and  others,  however,  the  strict  specificity  of 

^  Metchnikoff,  Ann.  de  I'inst.  Pasteur,  1898. 

2  Landsteiner,  Cent.  f.  Bakt.,  i,  25,  1899. 

^v.  Dungern,  Munch,  med.  Woch.,  1899. 

^  Neisser  und  Wechsberg,  Zeit.  f.  Hyg.,  xxxvi,  1901. 

^  Delezenne,  Ann.  de  Tinst.  Past.  1900;  Compt,  rend,  de  I'acad.  des  sci.   1900. 

•  Surmont,  Compt.  rend,  de  la  soc.  de  biol.,  1901. 
^  Bogart  et  Bernard,  ibid.,  1891. 

*  Ascoli  and  Figari,  Berl.  klin.  Woch.^  1902. 


202  INFECTION  AND  IMMUNITY 

nephrotoxin  could  not  be  upheld  and  the  subject  is  still  in  the  ex- 
perimental stage. 

Recent  experiments  by  Pearce  ^  suggest  that  at  least  a  part  of  the 
local  injury  to  organs  exerted  by  such  "cytotoxic"  sera  may  not  be 
due  to  a  specific  action  upon  the  organ  cells  so  much  as  upon  the 
hemaggiutinating  action  of  the  sera  causing  emboHsm  and  necrosis. 

It  is  a  fact  also  that  most  cytotoxic  sera  are  usually  hemolytic  as 
well.  It  is  not  easy  to  decide,  therefore,  how  much  of  the  action  upon 
the  organs  is  due  to  their  true  cytotoxic  properties  and  how  much  is 
attributable  to  the  concomitant  action  upon  blood  cells.  The  extrav- 
agant hopes  at  first  based  upon  cytotoxin  investigation,  especially  in 
regard  to  the  problem  of  malignant  tumors,  have  been  disappointed, 
and  much  is  still  obscure  in  regard  to  the  cytotoxins  which>  calls  for 
further  research. 

The  many  points  of  similarity  existing  between  bacterial  toxins  and 
digestive  ferments,  by  animal  inoculation,  suggested  to  several  observ- 
ers the  possibility  of  producing  antibodies  against  the  latter.  As  a 
result,  a  number  of  antiferments  have  been  obtained,  chief  among  which 
are  antilab  (Morgenroth  ^),  antipepsin  (Sachs  ^) ,  antisteapsin  (Schiitze  *), 
and  antilactase  (Schiitze). 

The  stimulation  of  antibody  formation  in  the  sera  of  animals  is  a 
consequence,  therefore,  of  the  injection  of  a  large  variety  of  substances — 
some  of  them  poisonous,  some  of  them  entirely  innocuous.  The  sub- 
stances possessing  this  power  have  been  conveniently  named  antigens  or 
antibody-producers  by  German  v/riters.  The  term  antigen — though  ety- 
mologically  wrong,  nevertheless  is  convenient  and  has  crept  into  general 
usage.  It  signifies  simply  a  substance  which  can  stimulate  the  pro- 
duction or  formation  of  an  antibody.  Such  substances,  so  far  as  is 
known,  belong  to  the  group  of  proteins  and  are  derivatives  of  animal  or 
plant  tissues.  Being  proteins,  all  antigens  are  colloids.  Recently,  how- 
ever, some  crystalloidal  substances  have  been  described  as  possessing 
antigenic  properties. 


1  Pearce,  Jour.  Exper.  Med.,  viii,  1906. 

2  Morgenroth,  Cent.  f.  Bakt.,  1899. 
^  Sachs,  Fort.  d.  Med.,  1902. 

*  Schiitze,  Deut.  med.  Woch.,  1904;  Zeit.  f.  Hyg.,  1905. 


CHAPTER   XIII 

TOXINS  AND  ANTITOXINS 

The  Toxin-Antitoxin  Reaction. — Apart  from  the  therapeutic  possi- 
biUties  disclosed  by  the  discovery  of  antitoxins,  new  light  of  inestimable 
value  was  thrown  by  these  observations  upon  the  biological  processes 
involved  in  immunization.  The  most  vital  problem,  of  course,  which 
immediately  thrust  itself  upon  all  workers  in  this  field  was  the  question 
as  to  the  nature  of  the  reaction  in  which  toxin  was  rendered  innocuous 
by  antitoxin. 

The  simplest  conception  of  this  process  would  be  an  actual  destruction 
of  the  toxin  by  its  specific  antitoxin,  and  it  is  not  unnatural,  therefore, 
that  this  was  the  view  which,  for  a  short  time,  found  favor  with  some 
observers.  Roux,  and  more  particularly  Buchner,^  however,  under  the 
sway  of  cellular  pathology,  advanced  the  opinion  that  the  antitoxins 
in  some  way  influenced  the  tissue  cells,  rendering  them  more  resistant 
against  the  toxins.  Antitoxin,  according  to  this  theor}^,  did  not  act 
directly  upon  toxin,  but  affected  it  indirectly  through  the  mediation 
of  tissue  cells.  Ehrlich,^  on  the  other  hand,  conceived  that  the  reac- 
tion of  toxin  and  antitoxin  was  a  direct  union,  analogous  to  the  chem- 
ical neutralization  of  an  acid  by  a  base — an  opinion  in  which  Behring 
soon  joined  him. 

The  conception  of  toxin  destruction  received  unanswerable  refuta- 
tion by  the  experiments  of  Calmette.^  This  observer,  working  with  snake 
poison,  found  that  the  poison  itself  (unlike  most  other  toxins)  possessed 
the  property  of  resisting  heat  even  to  100°  C,  while  its  specific  anti- 
toxin, like  other  antitoxins,  was  delicately  thermolabile.  He  noted, 
furthermore,  that  non-toxic  mixtures  of  the  two  substances,  when  sub- 
jected to  heat,  regained  their  toxic  properties.  The  natural  inference 
from  these  observations  could  only  be  that  the  toxin  in  the  original  mix- 
ture had  not  been  destroyed,  but  had  been  merely  inactivated  by  the 

^  Buchner,  "  Schutzimpfung/'etc,  in  Penzoldt  u.  Stinzing,  "Handbuch  d.  spez. 
Therap.  d.  Infektkrank./'  1894. 

^Ehrlich,  Deut.  med.  Woch.,  1891.        ^Calmette,  Ann.  de  Finst.  Past.,  1895. 

203 


204  INFECTION  AND  IMMUNITY 

presence  of  the  antitoxin,  and  again  set  free  after  destruction  of  the 
antitoxin  by  heat.  A  similar  observation,  made  soon  after  by  Wasser- 
mann  ^  in  the  case  of  pyocyaneus  toxin  and  antitoxin,  fully  supported 
the  results  of  Calmette. 

An  ingenious  proof  of  the  direct  action  of  antitoxin  upon  toxin 
was  obtained  by  Martin  and  Cherry.^  It  was  found  by  them  that  veiy 
dense  filters,  the  pores  of  which  had  been  filled  with  gelatin,  permitted 
toxin  to  pass  through  under  high  pressure,  while  the  presumably  larger 
antitoxin  molecule  was  held  back.  Through  such  filters  they  forced 
toxin-antitoxin  mixtures,  under  a  pressure  of  fifty  atmospheres,  at  vary- 
ing intervals  after  -mixing.  They  found  that,  if  filtered  inmiediately, 
all  the  toxin  in  the  mixtures  came  through,  but  that,  as  the  interval 
elapsing  between  mixing  and  filtration  was  prolonged,  less  and  less  toxin 
appeared  in  the  filtrate,  until,  finally,  two  hours  after  mixing,  no  toxin 
whatever  passed  through  the  filter.  Besides  demonstrating  the  direct 
action  of  antitoxin  upon  toxin,  this  work  of  Martin  and  Cherry  showed 
that  the  element  of  time  entered  into  the  toxin-antitoxin  reaction,  just 
as  it  enters  into  reactions  of  known  chemical  nature.  The  absolute  non- 
participation  of  the  living  tissue  cells  in  these  reactions  was  demonstrated 
by  Ehrlich  himself.  Kobert  and  Stillmarck  ^  had  shown  that  ricin  pos- 
sessed the  power  of  causing  the  red  blood  cells  of  defibrinated  blood  to 
agglutinate  in  solid  clumps,  a  reaction  which  could  easily  be  observed 
in  vitro.  EhrHch,'*  who  had  obtained  antiricin  in  1891  by  injecting 
rabbits  with  increasing  doses  of  ricin,  found  that  this  antibody  pos- 
sessed the  power  of  preventing  the  hemagglutinating  action  of  ricin 
in  the  test  tube.  By  a  series  of  quantitatively  graded  mixtures  of  ricin 
and. antiricin,  with  red  blood  cells  as  the  indicator  for  the  reaction,  he 
succeeded  in  proving  not  only  that  the  toxin-antitoxin  neutralization 
was  in  no  way  dependent  upon  the  living  animal  body,  but  that  definite 
quantitative  relations  existed  between  the  two  substances  entirely 
analogous  to  those  which,  according  to  the  law  of  multiple  proportions, 
govern  reactions  between  different  substances  of  known  chemical 
nature.  Similar  quantitative  results  were  subsequently  obtained  by 
Stephens  and  Myers  ^  for  cobra  poison  and  its  antitoxin,  by  Kossel  * 

1  Wassermann,  Zeit.  f.  Hyg.,  xxii,  1896. 

^Martin  and  Cherry,  Proc.  Royal  Soc,  London,  Ixiii,  1898, 

*  Kobert  und  Stillmarck,  Arb.  d.  phar.  Inst.  Dorpat, 

*  Ehrlich,  Fort.  d.  Med.,  1897. 
»  Stephens  and  Myers,  Jour,  of  Path,  and  Baet.,  1898. 

*  Kossel,  Berl.  klin.  Woch.,  1898. 


TOXINS  AND  ANTITOXINS  205 

for  the  toxic  eel  blood  serum,  and  by  Ehrlich  ^  for  the  hemolytic  tetanus 
poison  known  as  tetanolysin. 

The  introduction  of  the  test-tube  experiment  into  the  investigation 
of  these  reactions  permitted  of  much  more  exact  observations,  and  by 
this  means,  as  well  as  by  careful,  quantitatively  graded,  animal  experi- 
ments, the  further  facts  were  ascertained  that  toxin  and  antitoxin  com- 
bined more  speedily  in  concentrated  than  in  dilute  solutions,  and  that 
warmth  hastened,  while  cold  retarded,  the  reaction — observations^ 
which  in  every  way  seem  to  bear  out  Ehrlich's  conception  of  the  chemi- 
cal nature  of  the  process. 

Ehrlich's  Analysis  of  Diphtheria  Toxin. — Shortly  §tfter  the  discovery 
and  therapeutic  application  of  diphtheria  antitoxin,  it  became  apparent 
that  no  two  sera,  though  similarly  produced,  could  have  exactly  the 
same  protective  value.  It  was  necessary,  therefore,  to  establish  some 
measure  or  standard  by  which  the  approximate  strength  of  a  given  anti- 
toxin could  be  estimated.  Von  Behring  ^  attempted  to  do  this  for 
both  tetanus  and  diphtheria  antitoxins  by  determining  the  quantity  of 
immune  sera  which,  in  each  case,  was  needed  to  protect  a  guinea-pig  of 
known  weight  against  a  definite  dose  of  a  standard  poison.  He  ascer- 
tained the  quantity  of  standard  toxin-bouillon  which  would  suffice  to  kill 
a  guinea-pig  of  250  grams,  and  called  this  quantity  the  "toxin  unit.'' 
This  unit  was  later  more  exactlj'  limited  by  Ehrlich,  who,  considering 
the  element  of  time,  stated  it  as  the  quantity  sufficient  to  kill  a  guinea- 
pig  of  the  given  weight  in  from  four  to  five  days. 

Appropriating  the  terminology  of  chemical  titration,  v.  Behring 
spoke  of  a  toxin-bouillon  which  contained  one  hundred  such  toxin  units 
in  a  cubic  centimeter,  as  a  "normal  toxin  solution"  ("  DTN^  M^^""), 
and  designated  as  "normal  antitoxin-"  a  serum  capable  of  neutraliz- 
ing, cubic  centimeter  for  cubic  centimeter,  the  normal  poison.*  A  cubic 
centimeter  of  such  an  antitoxic  serum  was  sufficient,  therefore,  to  neu- 
tralize one  hundred  toxin  units,  and  was  spoken  of  as  an  "antitoxin 
unit."  In  the  experiments  of  v.  Behring,  toxin  and  antitoxin  had  been 
separately  injected.  Ehrlich  ^  improved  upon  this  method  by  mixing 
toxin  and  antitoxin  before  injection,  thereby  obviating  errors  arising 

»  Ehrlich,  Berl.  klin.  Woch.,  1898. 

«  Knorr,  Fort.  d.  Med.,  1897. 

»  V.  Behring,  Deut.  med.  Woch.,  1893. 

*  DTN  1  M250  signifies:  D,  Diphtheria;  TN^  Normal  Toxin  solution;  M«»,  Meer- 
schweinchen  or  guinea-pig  weighing  250  grams. 

*  Ehrlich,  Koesel  und  Wassermann,  Deut.  med.  Woch.,  1894. 


206  INFECTION  AND   IMMUNITY 

from  differences  which  may  have  existed  in  the  depth  of  injection  or 
rapidity  of  absorption. 

In  order,  however,  that  any  such  method  of  standardization  of  an- 
titoxin may  be  practically  applicable,  it  is  necessary  to  produce  either 
a  stable  toxin  or  an  unchangeable  antitoxin.  This  EhrHch  achieved  for 
antitoxin  by  drying  antitoxic  serum  in  vacuo  and  preserving  it  in  the 
dark,  at  a  low  temperature  and  in  the  presence  of  anhydrous  phosphoric 
acid.  By  the  use  of  such  a  stable  antitoxin,  various  toxins  may  be 
measured  and  other  antitoxic  sera  estimated  against  these. 

Given  thus  a  constant  antitoxin,  the  standardization  of  toxins  would 
be  a  comparatively  simple  matter  Avere  the  poison  obtainable  in  a  per- 
fectly pure  state.  Unfortunately  for  thp  ease  of  measurement,  how- 
ever, this  is  not  the  case.  The  problem  is  rendered  difficult  by  a  number 
of  compHcating  factors,  many  of  which  have  been  brought  to  light  by 
Ehrlich  ^  in  his  laborious  researches  into  the  quantitative  relationship 
between  the  two  reacting  bodies. 

As  previously  stated,  it  had  been  noted  by  Ehrlich  and  others  that 
toxin  solutions  would  deteriorate  with  time;   that  is,  a  toxin-bouillon 

^Toxop"hore\ 
<s^^       ^aplopKore) 


Body  Cell 

Fig.  55. — Toxin  and  Body  Cell. 

which  was  found  soon  after  production  to  contain,  say,  eighty  toxin 
units  in  each  cubic  centimeter,  would,  after  four  or  five  months,  be  found 
to  contain  but  forty  units  in  the  same  gross  quantity.  It  had  lost,  there- 
fore, in  this  case,  just  one-half  of  its  toxic  power.  In  spite  of  this  loss, 
however,  Ehrlich  found  that  such  bouillon  had  retained  its  fuU  original 
power  of  neutralizing  antitoxin.  If  the  reaction  was  purely  one  of 
chemical  neutralization,  there  seemed  to^be  but  one  explanation  of  this. 
The  toxin  molecule  must  contain  two  separate  atom  groups.  One  of 
these  must  possess  the  power  of  binding  antitoxin  and  be  stable;   this 

1  Ehrlich,  Klin.  Jahrbuch,  vi,  1897;   Deut.  med.  Woch.,  1898. 


TOXINS  AND  ANTITOXINS  207 

he  designates  as  the  "haptophore"  or  ''anchoring"  group.  The  other, 
the  one  by  which  the  toxin  molecule  exerts  its  deleterious  action,  must 
be  more  easily  changed  or  destroyed ;  this  he  calls  the  "  toxophore  " 
or  ''  poison  "  group.  In  the  altered  toxin-bouillon  in  which  a  part  of 
the  poisonous  action  has  been  lost  while  the  antitoxin-neutralizing  power 
is  intact,  the  toxophore  group  of  some  of  the  toxin  must  have  been 
changed  or  destroyed.     Such  altered  toxin  he  speaks  of  as  "toxoid.'^ 

In  support  of  this  hypothesis  and  for  the  purpose  of  perfecting  the 
methods  of  standardization,  Ehrlich  was  led  to  determine,  for  a  large 
variety  of  specimens  of  diphtheria  toxin,  the  precise  quantity,  in 
cubic  centimeters,  which  was  necessary  to  neutralize  exactly  one  unit 
of  his  standard  antitoxin.  This  he  accomplished  by  making  a  series  of 
toxin-antitoxin  mixtures,  in  each  of  which  the  quantity  of  antitoxin 
was  exactly  one  unit,  while  the  amount  of  toxin  was  gradually  increased. 
These  mixtures  were  injected  into  guinea-pigs  of  250  grams  weight. 
It  is  self-evident  that  in  such  an  experiment  the  mixtures  containinS 
the  smaller  quantities  of  toxin  would  have  no  effect  upon  the  guinea- 
pigs.  Soon,  however,  a  mixture  would  be  reached  in  which  toxin  would 
be  sufficiently  in  excess  of  antitoxin  to  produce  the  symptoms  of  slight 
poisoning,  as  evidenced  in  local  edema,  rise  of  temperature,  etc.  The 
largest  quantity  of  toxin  which  could  be  added  without  producing  such 
symptoms  was  then  regarded  as  exactly  neut*ralizing  one  antitoxin  unit. 
This  quantity  of  toxin  Ehrlich  speaks  of  as  "Limes  zero"  (Limes  = 
threshold)  or,  briefly,  "  L^." 

For  instance : 

One  antitoxin  unit  +  0.6  c.c.  toxin No  symptoms  of  poisoning. 

0.8  c.c " 

«         0.9  c.c "         " 

"  1.    c.c. "        "  "  " 

"  1.1  c.c Local  edema.    Paralysis  in  30  days. 

"  1.2  c.c Death  in  10  days. 

In  this  example,  L^,  therefore,  equals  1  c.c. 

It  is  obvious,  however,  that  because  of  the  great  difficulty  in  esti- 
mating the  very  slightest  evidences  of  toxic  action  in  guinea-pigs,  a 
more  exact  method  of  standardizing  the  poisons  against  antitoxin 
would  be  to  deteraiine  how  much  toxin  would  be  required  to  neu- 
tralize one  antitoxin  unit  and  still  be  sufficiently  in  excess  to  cause 
the  death  of  a  guinea-pig  of  250  grams  in  four  to  five  days .  This  would 
then  correspond  to  the  action  of  one  toxin  unit,  unmixed  with  antitoxin. 
A  priori  it  would  seem  that  this  value  (expressed  by  Ehrlich  as  "  Limes 


208  INFECTION  AND   IMMUNITY 

death  "  or  "  L+)  must  simply  be  L^  plus  one  toxin  unit.  This,  however, 
was  found  not  to  be  the  case.  Thus,  in  the  example  given,  in  which  T 
(the  toxin  unit — the  quantity  of  the  bouillon  kiUing  a  guinea-pig  of  2,50 
grams  in  four  to  five  days)  was  equal  to  O.Olc.c,  L^,  (the  quantity  of 
toxin  completely  neutralizing  one  antitoxin  unit)  was  found  to  be  1  c.c. 
or  100  T.  In  this  same  poison,  however,  L_^  (the  quantity  of  toxin  neces- 
sary both  to  neutralize  one  antitoxin  unit  and  yet  to  be  sufficiently  in 
excess  of  neutralization  to  kill  a  guinea-pig  of  250  grams  in  four  or  five 
days)  was  not  found  to  be  merely  L^  +  IT;  but  on  actual  experi- 
ment proved  to  be  L^  +  101  T. 

Expressed  graphically,  the  conditions  may  be  stated  as  follows: 

T=  .01  c.c.  of  the  toxin  bouillon. 

L  +  (neutral,  of  1  antitox.  unit  yet  killing  1  pig)  =  2.01  c.c.  or  201  T. 

Lq    (complete  neutral,  of  1  antitox.  unit)  =  1.      c.c.  or  100  T. 

Difference  =  1.01  c.c.  or  101  T. 

Ehrlich,  at  first,  endeavored  to  explain  this  surprising  phenomenon 
on  the  basis  of  toxoids.  He  argued  that  the  toxoids  formed  by  de- 
terioration of  toxin  might  be  conceived  as  possessing  three  different 
degrees  of  affinity  for  antitoxin.  If  their  affinity  for  antitoxin  were 
equal  to,  or  more  marked  than,  that  of  the  toxin  itself,  they  could  have 
no  influence  upon  the  dose  L^  .  If,  however,  their  affinity  for  antitoxin 
were  weaker  than  that  of  to!xin,  each  fresh  toxin  unit  added  to  the  dose 
Lq  would,  first  uniting  with  antitoxin,  replace  a  corresponding  quan- 
tity of  these  nontoxic  substances  of  weaker  affinity,  and  L^  would 
be  reached  only  after  all  of  these  "  epitoxoids,"  as  Ehrlich  called  them, 
had  been  replaced,  and  toxin  became  free  in  the  mixture. 
Thus,  in  analyzing  our  example,  we  have: 

100   tox.-antitox.  +  100   epitox.-antitox.  =  Lq  ; 
add  1  T,  and  we  have  101  tox.-antitox.  +  99  epitoxoid-antitoxin  +  1  epitoxoid  free; 
add  101  T  and  we  have  200  toxin-antitoxin  +  100  epitoxoid  free  +1  T  free  =  L^. 

Two  facts,  however,  led  Ehrlich  to  abandon  the  opinion  that  epi- 
toxoid was  merely  a  variety  of  toxoid.  He  found,  in  the  first  place, 
that  the  stated  relations  between  L^  and  L^  were  true  for  perfectly, 
fresh  toxin-bouillon  in  which  little  or  no  deterioration  had  taken  place. 
He  observed,  furthermore,  that  in  old,  altered  toxin  bouillon,  while 
T  was  very  much  affected,  the  quantity  needed  to  kill  a  pig  con- 
stantly increasing,  and    the  number  of    actual  fatal  doses  in  L^  cod- 


TOXINS  AND  ANTITOXINS  209 

stantly  decreasing  (by  reason  of  toxoid  formation),  L+  remained 
practically  unchanged. 

Simply  stated,  this  means  that  the  epitoxoids  or  substances  which 
have  weaker  affinity  for  antitoxin  than  toxin  itself  are  already  present 
in  fresh  bouillon  and  are  not  increased  with  time.  For  this  reason, 
Ehrlich  has  separated  these  substances  from  toxoids.  He  calls  them  "  tox- 
on"  and  beheves  them  to  be,  Uke  toxin,  primary  secretory  products  of 
the  diphtheria  bacilli.  The  toxoids  themselves,  Ehrlich  believes,  are  of 
two  kinds,  those  with  a  stronger  affinity  for  antitoxin  than  toxin  it- 
self (protoxoids) ,  and  those  whose  affinity  for  antitoxin  is  equal  to  that 
of  toxin.    These  latter  he  calls  "syntoxoids.'' 

The  toxon  (epitoxoid  originally),  as  Ehrlich  believes,  has  a  hapto- 
phore  or  "binding"  group  similar  to  that  of  toxin,  but  a  different 
toxophore  or  "poisoning"  group.  Qualitatively  it  has  been  shown 
to  differ  from  toxin  in  that,  lacking  the  power  to  produce  acute  symp- 
toms, it  causes  gradual  emaciation  and  paresis  in  animals. 

That  this  difference  in  the  poisonous  action  of  toxin  and  toxon 
is  not  merely  a  quantitative  difference,  referable  to  small  quantities  of 
toxin,  was  proved  by  Dreyer  and  Madsen,^  who  showed  that  if  they  made 
a  toxin-antitoxin  mixture  in  which  after  injection  the  only  evidence  of 
incomplete  neutralization  lay  in  the  emaciation  and  final  paralysis  of 
the  test  animals,  the  quantity  of  such  a  mixture  could  be  increased 
five-  and  tenfold,  without  producing  the  true  toxin  symptoms  in  ani- 
mals. These  authors,  too,  claim  to  have  been  able  to  immunize  against 
toxin  with  such  mixtures,  thereby  proving  the  identity  of  the  haptophore 
groups  of  the  two  substances.  The  importance  of  this  observation  will 
become  more  evident  in  connection  with  the  section  on  the  "side- 
chain  theory." 

Method  of  Partial  Absorption  of  Toxin. — Ehrfich  ^  has  gathered 
more  exact  data  in  support  of  his  views  from  what  he  terms  the  "  Method 
of  Partial  Absorption  "  of  toxin  by  antitoxin. 

In  order  to  understand  this  method  clearly,  it  is  necessary  to  re- 
member that  Ehrlich  ^  believes  the  union  of  toxin  with  antitoxin  to 
take  place  according  to  the  chemical  laws  of  valency.  Just  as  in  HjO 
oxygen  has  an  atomic  valency  of  2  for  hydrogen,  so,  in  the  fully 
neutralized  toxin-antitoxin  compound,  he  believes  antitoxin  to  have  a 


1  Dreyer  und  Madsen,  Zeit.  f.  Hyg.,  xxxvii,  1901. 
#2  Ehrlich,  " Gesammelte  Arbeiten  zur  Immunitatsforsch.,"  Berlin,  1904. 
8  Ehrlich,  Deut.  med.  Woch.,  1898. 
15 


210  INFECTION     AND  IMMUNITY 

valency  of  200  for  toxin.  It  would  require,  according  to  this,  200  T 
or  toxin  molecules  to  satisfy  the  affinities  of  one  antitoxin  molecule.* 

This  behef  is  based  upon  the  following  consideration:  In  determining 
the  Lq  dose,  or  fully  neutrahzed  toxin-antitoxin  union,  EhrKch,  as  well 
as  Madsen,  found  that  the  number  of  T  units  contained  in  such  a 
dose  was  almost  regularly  a  factor  of  one  hundred,  recurring  again 
and  again  as  25,  33,  50,  75,  etc.  This  pointed  to  more  or  less  regularity 
in  the  deterioration  of  toxin  into  toxoid,  and  to  a  more  or  less  regular 
relation  of  toxin  to  toxon.  Now,  as  we  have  seen  before,  if  we  could 
procure  a  perfectly  pure  toxin,  the  L^  dose  plus  one  toxin  unit  would 
give  us  the  L+  dose;  that  is,  one  toxin  unit  in  excess  of  full  neutraliza- 
tion would  suffice  to  kill  a  guinea-pig  of  250  grams  in  four  to  five  days. 
Since  a  perfectly  pure  toxin,  however,  has  not  been  obtainable  up  to  the 
present  time,  it  is  clear  that  the  number  of  pure  toxin  bonds  contained 
in  L^  must  be  less  than  the  actual  number  of  neutralizing  units  in  the 
combination,  a  part  of  the  antitoxin  being  bound  by  toxon  and  1?oxoid. 
The  actual  values  obtained  for  the  number  of  T  units  in  L^.  has 
never  exceeded  200,  and  has  usually  been  more  than  100,  the  highest 
value  ascertained  by  Madsen  being  160.  Given,  therefore,  a  combining 
v,alue  which,  being  a  multiple  of  one  hundred,  is  often  more  than  one 
hundred,  but  in  an  obviously  impure  state  has  never  reached  200,  it  is 
most  likely  that  200  represents  the  actual  value  sought  for. 

Assuming,  therefore,  upon  the  foregoing  considerations,  that  the 
valency  of  antitoxin  for  toxin  is  200,  Ehrlich  carries  out  his  experi- 
ments in  the  following  way: 

Given  a  toxin,  the  unit  (T)  of  which  is  0.024  c.c,  he  first  deter- 
mines the  L+  dose  which,  tested  against  the  standard  antitoxin  unit,  in 
this  case  is  2.05  c.c.  But  2.05  c.c.  =  85  T.  (or  2.05  ^  .024)  units. 
By  mixing  the  L+  dose  of  toxin  and  antitoxin  in  such  a  way  that  the 
quantity  of  antitoxin  is  gradually  increased,  while  the  toxin  remains 
always  L+,  and  determining  upon  animals  the  amount  of  free  toxin 
contained  in  each  mixture,  the  following  table  may  be  constructed:^ 

0  antitox.  unit  representing  0  valencies +  L+  =  85    free  T  units. 

.1        "          "            "  20        "        +L+  =  85      "  "     " 

.25     "          "            "  50        "        +L+  =  60      "  "     " 

.8       "          "            "  160       "        +L+  =  10      "  "     " 

.9        "          "            "  180        "        +L+  =     3.5  "  "     " 

*  1  Ehrlich,  "  Schlussbetrachtungen,"  Nothnager^  System.  0 

2  Example  taken  from  Ehrlich,  Deut.  med.  Woch.,  1898 


TOXINS  AND  ANTITOXINS 


211 


It  is  plain  that  the  substances  with  the  strongest  affinity  for  antitoxin 
must  be  bound  first  by  the  antitoxin.  This  does  not  diminish  the  toxic 
value  of  the  mixture;  and  these  are  the  pro  toxoids.  Next  are  bound 
syntoxoids  and  toxins,  and,  finally,  the  toxons.  It  is  plain  that,  by 
this  method,  the  constitution  of  any  given  toxin  may  be  ascertained, 
and  Ehrlich  has  constructed,  on  the  basis  of  these  observations,  what  he 
terms  his  toxin  spectrum.  Minor  differences  of  toxicity  and  affinity  for 
the  antibody  have  caused  him,  by  the  partial  saturation  method  de- 
scribed, still  further  to  divide  toxin  into  proto-,  deutero-,  and  trito-toxin. 
His  spectra  graphically  describe  the  constitution  of  any  given  toxic 
bouillon  and  trace  its  deterioration  as  follows: 


•   fo  io  Jo  io  30  so  70 .  80  90  m  m  i20 130  in  mf'60  ^7e?fSff  i9o  !09 


f^2. 


Prvtotoxlns  DeuUro-        Tritotoxins 
toxins 


Prototaxoids  cC  toxoids  cc     TYitotoxoids  aC 


ProCotoxins /3  DeiUero-    Triiotaxlns  /3 
toxiru/^ 


Ehrlich's  opinion  as  to  the  constitution  of  toxin  is  by  no  means 
fully  accepted.     Arrhenius,^  the  great  physical  chemist,  and  Madsen, 


Arrlwnius\xvAMad$m,  Zeit.  f.physik.Chem.,  1902;  Festschrift,  Kopenhagen,  1902. 


212  INFECTION  AND  IMMUNITY 

a  bacteriologist  who  was  once  a  pupil  of  Ehriich,  have  recently  opposed 
Ehriich^s  theory  on  grounds  of  physical  chemistry. 

Modem  theories  of  solution  maintain  that  substances  in  solution 
are  broken  up  into  their  atoms  or  atom-groups,  known  as  ions.  Thus, 
NaCl  in  solution  would  be  "  dissociated  "  into  its  Na  ion  and  its  CI  ion, 
the  completeness  of  the  dissociation  depending  upon  the  concentra- 
tion of  the  solution.  A  solution  of  NaCl,  therefore,  contains,  according 
to  this  view,  three  substances,  NaCl  undissociated  and  free  ions  of  Na 
and  CI,  the  relative  quantities  of  the  three  present  in  any  given  solution 
being  calculable,  and  depend  upon  a  law  known  as  the  law  of  mass- 
action  of  Guldberg  and  Waage.  These  free  ions  are  the  elements,  there- 
fore, which  are  active  in  the  formation  of  further  chemical  combination. 
When  a  strong  acid,  in  solution,  acts  upon  a  base,  say  HCl  upon  am- 
monia (NHg),  strong  acid  having  the  property  of  quite  complete  dis- 
sociation in  relatively  concentrated  solutions,  little  or  no  ammonia 
would  remain  unbound.  A  weak  acid,  like  boric  acid,  however,  not  being 
as  completely  dissociated,  would  leave  some  ammonia  uncombined  even 
after  more  than  quantitatively  sufficient  boric  acid  had  been  added. 
Arrhenius  and  Madsen,  on  the  basis  of  careful  researches  into  the  re- 
action between  tetanolysin  and  its  antibody,  believe  that  toxin  and  anti- 
toxin possess  weak  chemical  avidity  for  each  other,  their  interaction 
being  comparable  to  that  taking  place  between  a  weak  acid  and  a  base. 
Toxin-antitoxin  solutions,  therefore,  would  contain  the  neutral  com- 
pound, but  at  the  same  time  uncombined  toxin  and  antitoxin.  The 
qualities  which  Ehriich  ascribes  to  toxon,  they  believe,  are  due  to 
the  unbound  toxin  present  in  such  mixtures.  In  careful  studies 
in  which  they  inhibited  the  hemolytic  action  of  ammonia  by  gradual 
addition  of  boric  acid,  they  were  able  to  show  complete  parallelism 
between  the  conditions  governing  this  neutralization  and  those  con- 
cerned in  their  tetanus  experiments.  Their  explanation  has  the 
advantage  of  extreme  simplicity  over  that  of  Ehriich,  but  since  the 
differences  of  opinion  are  now  the  subject  of  active  experimental 
controversy,  a  critical  discussion  must  rest  until  further  facts  are 
revealed. 

The  Side-Chain  Theory. — We  have  seen  that  the  extensive  researches 
of  Ehriich  into  the  nature  of  the  toxin-antitoxin  reaction  led  him  to 
believe  that  the  two  bodies  underwent  chemical  union,  forming  a  neu- 
tral compound.  The  strictly  specific  character  of  such  reactions,  further^ 
more,  diphtheria  antitoxin  binding  only  diphtheria  toxin,  tetanus 
antitoxin  only  tetanus  toxin,  etc.,  led  him  to  assume  that  the  chemical 


TOXINS  AND  ANTITOXINS  213 

affinity  between  each  antibody  and  its  respective  antigen  depended 
upon  definite  atom  groups  contained  in  each. 

Ehrlich  ^  had,  in  1885,  published  a  treatise  in  which  he  discussed 
the  manner  of  cell-nutrition  and  advanced  the  opinion  that  in  order  to 
nourish  a  cell,  the  nutritive  substance  must  enter  directly  into  chemical 
combination  with  some  elements  of  the  cell  protoplasm.  The  great 
number  and  variety  of  chemical  substances  which  act  as  nutriment  led 
him  to  believe  that  the  highly  complex  protoplasmic  molecules  of 
cells  were  made  up  of  a  central  atom-group  (Leistungs-Kern)  upon 
which  depended  the  specialized  activities  of  the  cell,  and  a  multi- 
plicity of  side  chains  (a  term  borrowed  from  the  chemistry  of  the 
benzol  group),  by  means  of  which  the  cell  entered  into  chemical  relation 
with  food  and  other  substances  brought  to  it  by  the  circulation.  If 
we  illustrate  graphically  by  the  chemical  conception  from  which  the 
term  side  chain  was  borrowed,  in  salicylic  acid,  the  formula  given,  the 

OH 

I 
C 

/\ 
H— C       C— COOH 

I         I 
H— C       C— H 

\y 

C 

I 
H 

benzol  ring  represents  the  "Leistungs-Kern,''  or  radicle,  while  OOOH 
and  OH  are  side  chains  by  means  of  which  a  variety  of  other  substances 
may  be  brought  into  relation  with  the  '^radicle,"  for  instance,  as  in 
methyl  salicylate. 

OH 

COoCHo 


Just  as  nutritious  substances  are  thus  brought  into  workable  re- 
lation with  the  cell  by  means  of  the  atom-groups  corresponding  to  side 
chains,  so  Ehrlich  believes  toxins  exert  their  deleterious  action  only 
because  the  cells  possess  side  chains  by  means  of  which  the  toxin  can  be 
chemically  bound.  These  side  chains,  Ehrlich  in  his  later  w^ork  calls 
"receptors."     The  receptors  or  side  chains  present   in  the  cells  and 

1  Ehrlich,  "  Das  Sauerstoffbediirfmss  des  Organismus,"  Berlin,  1885. 


214  INFECTION  AND  IMMUNITY 

possessing  by  chance  specific  affinity  for  a  given  toxin,  are,  by  their 
union  with  toxin,  rendered  useless  for  their  normal  physiological  func- 
tion. By  the  normal  reparative  mechanism  of  the  body  these  recep- 
tors are  probably  cast  off  and  regenerated.  Regenerative  processes  of 
the  body,  however,  do  not,  as  a  rule,  stop  at  simple  replacement  of  lost 
elements,  but,  according  to  the  hypothesis  of  Weigert,^  usually  tend  to 

overcompensation.     The  receptors  eliminated 
!i\\\l!llll/////^^To-3cin.  by  toxin  absorption  are  not,  therefore,  simply 

reproduced   in  the   same  quantity  in  which 
they  are  lost,  but  are  reproduced  in  excess  of 
..^nUipxin     the  simple  physiological  needs  of  the  cell. 
Continuous  and  increasing  dosage  with  the 
poison,    consequently,   soon    leads    to    such 
Fig   se-'IbxiN  and         excessive  production  of    the    particular   re- 
Antitoxin.  ceptive  atom-groups  that  the  cells  involved 

in  the  process  become  overstocked  and  cast 
them  off  to  circulate  freely  in  the  blood.  These  freely  circulating  re- 
ceptors— atom-groups  with  specific  affinity  for  the  toxins  used  in  their 
production — represent  the  antitoxins.  These,  by  uniting  with  the 
poison  before  it  can  reach  the  sensitive  cells,  prevent  its  deleterious 
action.     (Fig.  56.) 

The  theory  of  Ehrlich,  in  brief,  then,  depends  upon  the  assumptions 
that  toxin  and  antitoxin  enter  into  chemical  union,  that  each  toxin 
possesses  a  specific  atom-group  by  means  of  which  it  is  bound  to  a  pre- 
existing side  chain  of  the  affected  cell,  and  that  these  side  chains,  in  ac- 
cordance with  Weigert's  law,  under  the  influence  of  repeate-d  toxin  stimu- 
lation, are  eventually  overproduced  and  cast  off  by  the  cell  into  the 
circulation. 

It  stands  to  reason  that  this  theoretical  conception  would  be  vastly 
strengthened  were  it  possilSle  to  show  that  such  receptors  or  toxin- 
binding  atom-groups  actually  pre-existed  in  the  animal  body,  and  such 
support  was  indeed  given  by  the  experiments  of  Wassermann  and  Taka- 
ki.^  These  observers  succeeded  in  showing  that  tetanus  toxin  could  be 
rendered  innocuous  if,  before  injection  into  animals,  it  was  thoroughly 
mixed  with  a  sufficient  quantity  of  the  fresh  brain  substance  of  guinea- 
pigs.    Similar  observations  were  independently  made  by  Asakawa,""*  and 


1  Weigert,  Verhandl.  d.  Ges.  Deutsch.  Naturf.  u.  Aerzte,  Frankfurt,  1896. 

2  Wassermann  und  Tdkdki,  Berl.  klin.  Woch.,  1898 
5  A-sakawa,  Cent.  f.  Bakt.,  1898. 


TOXINS  AND  ANTITOXINS  215 

variously  confirmed.  Kempner  and  Schepilewsky  ^  showed  a  similar 
relation  to  exist  between  brain  tissue  and  botulismus  toxin,  and  Myers  ^ 
brought  proof  of  analogous  conditions  in  the  case  of  suprarenal  tissue 
and  cobra  poison. 

In  the  discussion  of  Ehrlich's  toxin  analysis,  we  have  seen  that  he 
accounted  for  variations  in  the  quantitative  relations  by  the  existence 
of  toxoids  and  toxons.  He  explained  the  striking  fact  that  toxoids  had 
lost  their  poisonous  nature  and  yet  retained  full  powers  to  neutralize 
antitoxin  by  the  assumption  that  toxin  was  made  up  of  two  separate 
atom-groups;  one,  the  haptophore  group  which  possessed  the  specific 
affinity  for  the  antitoxin  or  cell  receptor;  the  other,  the  toxophore 
group  by  means  of  which  the  actually  harmful  effects  were  produced. 
The  haptophore  groups  of  all  three  of  these  substances,  toxin,  toxoid, 
and  toxon,  by  virtue  of  their  antitoxin-binding  power,  he  assumed  to  be 
alike;  in  toxoid,  the  toxophore  group  has  been  destroyed  or  altered; 
in  toxon,  the  toxophore  group  is  qualitatively  different  from  that  of 
toxin.  The  haptophore  group,  however,  being  alone  concerned  in  neu- 
tralizing receptors,  all  three  of  these  substances  should,  if  Ehrlich's 
theory  is  to  be  tenable,  produce  antitoxin.  Dreyer  and  Madsen,'^  ac- 
cordingly, actually  succeeded  in  producing  diphtheria  antitoxin  by  im- 
munization with  toxon.  Attempts  to  produce  antitoxin  with  toxoids 
have  succeeded  in  the  hands  of  Ehrlich  and  others.  Such  experiments 
have  not,  however,  been  always  successful,  a  notable  failure  being  that 
of  Bruck.^  On  the  basis  of  such  negative  results  the  theory  was  advanced 
by  Wassermann  that  overproduction  of  receptors  was  stimulated  by 
the  irritation  (Zellreiz)  produced  by  the  toxophore  group^a  stimula- 
tion not  present  in  the  case  of  toxoids. 


1  Kempner  und  Schepilewsky,  Zeit,  f.  Hyg.,  1898. 

2  Myers,  Cent.  f.  Bakt.,  i,  1899.  a  Dreyer  iind  Madsen,  Zeit.  f.  Hyg.,  1901. 
*  Bruck,  Zeit.  f.  Hyg.,  1904. 


CHAPTER    XIV 
PRODUCTION  AND  TESTING   OF  ANTITOXINS 

DIPHTHERIA   ANTITOXIN 

In  spite  of  the  great  advances  in  our  theoretical  knowledge  of  anti- 
bodies, gained  during  the  last  three  decades,  extensive  therapeutic 
application  has  been  made  of  the  antitoxins  only.  Pre-eminent  among 
these  from  a  practical  point  of  view  are  the  antitoxins  against  diphtheria 
and  tetanus  toxin.  For  diphtheria,  careful  statistical  studies  have 
demonstrated,  beyond  doubt,  the  therapeutic  value  of  the  serum 
treatment.  Biggs  and  Guerard,  in  a  general  statistical  review,  ar- 
rived at  the  conclusion  that  the  death  rate  of  diphtheria  had  been  re- 
duced fifty  per  cent  by  the  use  of  antitoxin.  Approximately  the  same 
estimate  is  made  by  Dieudonne^  who  studied  almost  10,000  treated  cases. 

Production  of  Diphtheria  Antitoxin. — The  methods  for  producing 
diphtheria  antitoxin  vary  only  in  minor  technical  details.  The  first 
requisite  for  successful  antitoxin  production  is  the  possession  of  a  strong 
toxin.  The  various  means  of  obtaining  this  are  outlined  in  the  section 
on  diphtheria  toxin.  The  toxin  used  should  be  of  such  potency  that 
less  than  0.01.  cc.  will  kill  a  guinea-pig  of  250  grams  weight  in  four 
to  five  days.^ 

For  experimental  purposes,  goats  or  sheep  may  be  used  for  immuniza- 
tion; for  antitoxin  production  on  a  large  scale,  horses  have  been  found 
to  be  the  most  useful  animals.  The  horses  should  be  young,  four  to 
six  years  old,  vigorous,  and  healthy.  It  is  advisable  that  they  be  sub- 
jected to  the  mallein  test  to  exclude  possible  infection  with  glanders. 

The  toxin  injections  are  made  subcutaneously.  Because  of  the  dif- 
ferences in  susceptibility  noted  in  various  horses,  it  is  advisable  that  the 
first  doses  of  toxin  should  be  either  very  small  or  weakened  by  chemicals 
or  heat,  or  combined  with  antitoxin.  In  the  Pasteur  Institute  in  Paris, 
the  small  initial  dose  of  toxin  (0.5  cc.)  is  mixed  before  injection  with 
an  equal  quantity  of  LugoFs  solution  (iodin-potassium  iodid  solution). 

1  Dieudonne,  Arb.  a.  d.  kais.  Gesundheitsamt,  1895  and  1897. 

2  Park,  "  Pathog.  Bacteria  and  Protozoa,"  N.  Y.,  1908. 

216 


PRODUCTION  AND  TESTING  OF  ANTITOXINS  217 

Park  *  advises  an  initial  dose  of  5,000  toxin  units  (about  20  c.c.  of 
toxin)  combined  with  100  units  of  antitoxin.  The  same  amount  is  given 
with  the  second  and  third  doses  of  toxin.  The  intervals  are  from  five 
days  to  a  week,  determined  by  complete  subsidence  of  the  reaction  (tem- 
perature). The  doses  are  increased  until,  at  the  end  of  two  or  three 
months,  more  than  ten  times  the  original  dose  is  given  (50,000  units). 

Horses  vary  greatly  in  the  strength  of  antitoxin  which  they  will  pro- 
duce. At  the  end  of  three  or  four  months  in  favorable  animals  one 
cubic  centimeter  of  serum  may  contain  250  to  800  antitoxin  units.  Fur- 
ther immunization  will  often  increase  the  antitoxin  output  to  1,000  and 
more  units  to  the  cubic  centimeter  of  serum.  Park  states  that  none  of 
the  horses  used  by  him  has  ever  yielded  2,000  units  to  the  cubic  centi- 
meter. The  same  writer  advises  a  three  months'  period  of  rest  from 
immunization  at  the  end  of  every  nine  months.  Given  such  resting 
periods,  some  horses  have  continued  to  furnish  high-grade  antitoxin  for 
from  two  to  four  years. 

In  order  to  obtain  serum  from  horses,  a  sharp  cannula  is  introduced 
into  the  jugular  vein.  After  leading  the  horse  into  a  specially  con- 
structed stall,  its  head  is  slightly  deflected  and  pressure  is  made  upon  the 
jugular  vein  below  the  point  into  which  the  needle  is  to  be  plunged. 
Compression  can  also  be  made  by  surrounding  the  neck  of  the  horse  close 
to  the  shoulders  with  a  leather  strap  over  a  pad  laid  directly  upon 
the  vein.  The  vein  becomes  visible  along  the  lower  margin  of  the  neck 
in  a  line  running  from  the  angle  of  the  jaw  to  the  edge  of  the  scapula. 
The  skin,  of  course,  is  previously  shaved  and  sterilized.  The  cannula 
is  then  plunged  into  the  vein,  either  with  or  without  previous  incision 
through  the  skin,  and,  through  a  sterile  rubber  tube,  the  blood  is  al- 
lowed to  flow  into  high  glass  cylinders  or  slanted  Erlenmeyer  flasks. 
In  this  way,  large  quantities  of  blood  may  be  obtained  and,  according 
to  Kretz,2  as  much  as  six  liters  may  be  taken  at  a  time  at  intervals  of 
a  month,  without  injuring  the  animal.  Ligature  of  the  vein  after 
bleeding  is  unnecessary. 

The  cylinders  and  flasks  are  allowed  to  stand  for  two  or  three  days 
at  or  below  10°  C.  At  the  end  of  this  time,  the  serum  may  be  pipetted 
or  siphoned  away  from  the  clot  and  stored  in  the  refrigerator.  In  order 
to  diminish  the  chances  of  contamination,  five-tenths  per  cent  of  car- 
bolic acid  or  four-tenths  per  cent  of  tri-cresol  may  be  added. 

1  Park,  loc.  cit.,  p.  212. 

^  Kretz',  in  "Handb.  der  Techn.  u.  Meth.  d.  Immun.,"  Kraus  and  Levaditi,  vol.  ii, 
1908. 


218  INFECTION  AND  IMMUNITY 

Antitoxin  is  fairly  stable  and  if  kept  in  a  cool,  dark  place,  may  re- 
main active,  with  but  slight  deterioration,  for  as  long  as  a  year.  Kept 
in  a  dry  state,  in  vacuo,  over  anhydrous  phosphoric  acid,  by  the  method 
of  Ehrlich,  it  retains  its  strength  indefinitely. 

Standardization. — ^Antitoxin  units  being  measured  in  terms  of  toxin, 
uniformity  of  measurement  necessitates  the  possession  by  the  various 
laboratories  of  a  uniform  toxin.  Antitoxin  being  more  stable  than 
toxin,  uniformity  of  toxin  is  obtained  by  means  of  a  standard  antitoxin 
distributed  from  a  central  laboratory.  This  was  first  done  by  Ehrlich 
in  Germany,  and  is  now  done  for  the  United  States  by  the  Public  Health 
and  Marine  Hospital  Service  laboratories.  Bottles  of  the  distributed 
antitoxin  are  marked  with  the  number  of  units  contained  in  each  c.c. 
Dilutions  of  this  are  mixed  with  varying  quantities  of  the  toxin  to  be 
tested,  the  mixtures  are  allowed  to  stand  for  15  minutes  to  permit  union 
of  the  two  elements,  and  injections  into  guinea-pigs  of  250  grams  weight 
are  made.  Thus  the  L4.  dose  of  the  toxin  is  determined.  (The  L+  dose 
[p.  208]  is  the  quantity  of  poison  not  only  sufficient  to  neutralize  one 
antitoxin  unit,^  but  to  contain  an  excess  beyond  this  sufficient  to  kill  a 
guinea-pig  of  250  grams  in  4  to  5  days.  L4.  is  chosen  rather  than  Lo,  the 
simple  neutralizing  dose,  because  of  the  difference  between  toxins  in 
their  contents  of  toxoid  and  toxon.^) 

The  L+  dose  of  the  toxin  having  thus  been  determined,  this  quantity 
is  mixed  with  varying  dilutions  of  the  unknown  antitoxin.^  Thus,  given 
an  antitoxin  in  which  300  to  400  units  to  the  c.c.  are  suspected,  dilutions 
of  1  :  200,  1  :  250,  1  :  300,  etc.,  are  made.  One  c.c.  of  each  of  these  is 
mixed  with  the  L4-  dose  of  the  toxin,  and  the  mixtures  are  injected  into 
guinea-pigs  of  about  250  grams.  If  the  guinea-pig  receiving  L+  plus 
the  1  :  250  dilution  lives  and  the  one  receiving  L4.  plus  the  1  :  300  dilu- 
tion dies  in  the  given  time,  we  know  that  the  unit  sought  must  lie  be- 
tween these  two  values,  and  further  similar  experiments  will  easily 
limit  it  more  exactly.     The  possibility  of  error  in  carrying  out  such 


^The  older  definition  of  a  unit  of  diphtheria  antitoxin  is  the  quantity  of  anti- 
toxin sufficient  to  protect  a  guinea-pig  of  250  grams  against  100  times  the  fatal  dose 
of  diphtheria  toxin.  This,  however,  holds  true  only  if  we  are  dealing  with  normal 
toxins  and  antitoxins  as  at  first  devised  by  Behring.  In  the  conditions  under  which 
the  measurements  are  made  at  present,  however,  this  definition  must  be  revised  as 
follows:  A  unit  of  antitoxin  is  that  amount  of  antitoxin  which  will  save  the  life  of  a 
guinea-pig  if  injected  together  with  an  L4.  dose  of  the  toxin. 

^  Madsen,  in  Kraus  u.  Levaditi,  "Handbuch,"  etc.,  1907. 

3  Donitz,  "Die  Werthbem.  der  Heilsera,"  in  KoUe  u.  Wassermann. 


PRODUCTION  AND  TESTING  OP  ANTITOXINS  ^19 

measurement  is  much  diminished  by  the  use  of  larger  quantities  of  dilu- 
tions higher  than  those  given.      Four  e.c.  is  the  volume  usually  injected. 

Since  1902,  the  production  and  sale  of  diphtheria  antitoxin  has  been 
regulated  by  law  in  the  United  States.  From  time  to  time,  antitoxin  is 
bought  in  the  open  market  and  examined  at  the  hygienic  laboratories  of 
the  United  States  Public  Health  and  Marine  Hospital  Service.  Anti- 
toxic serum  which  contains  less  than  two  hundred  units  to  each  cubic 
centimeter  is  not  permitted  upon  the  market. 

In  a  previous  section  we  have  seen  that  Hiss  and  Atkinson  ^  and 
others  have  shown  an  increase  in  the  globulin  contents  of  blood  serum 
of  immunized  animals.  It  has  been  shown,  furthermore,  that  the  pre- 
cipitation of  such  serum  with  ammonium  gulphate  carried  down  in  the 
globulin  precipitate  all  the  antitoxic  substances  contained  in  the  serum. 
Upon  a  basis  of  globulin  precipitation,  Gibson  ^  has  recently  perfected  a 
method  of  concentrating  and  purifying  diphtheria  antitoxin  for  thera- 
peutic use.  This  procedure,  as  carried  out  at  the  New  York  Depart- 
ment of  Health,  is,  in  principle,  as  follows: 

The  serum,  as  taken  from  the  horse,  is  heated  to  56°  C.  for  twelve 
hours.  This  converts  about  half  of  the  pseudoglobulin  into  euglobulin, 
the  antitoxin  remaining  in  the  pseudoglobulin  fraction.^  It  is  then  * 
precipitated  with  an  equal  volume  of  a  saturated  ammonium  sulphate 
solution.  After  two  hours,  the  precipitate  is  caught  in  a  filter  and 
redissolved  in  a  quantity  of  water  corresponding  to  the  original  quantity 
of  serum.  After  filtration,  this  solution  is  again  precipitated  with 
saturated  ammonium  sulphate  solution  and  the  precipitate  again  fil- 
tered off.  The  precipitate  is  then  treated  with  a  saturated  solution  of 
sodium  chloride  of  double  the  volume  of  the  original  serum.  This  is 
allowed  to  stand  for  about  twelve  hours.  At  the  end  of  this  time  the 
antitoxin-containing  globulin  is  in  solution  and  is  pipetted  away  from 
the  precipitate  and  filtered.  This  salt-solution  extract  is  then  pre- 
cipitated with  twenty-five  hundredths  per  cent  acetic  acid.  The  re- 
sulting precipitate  of  globulin  is  thoroughly  dried  by  pressure  between 
filter  papers  and  placed  in  a  parchment  dialyzer.  Dialysis  with  run- 
ning water  is  continued  for  seven  to  eight  days,  after  neutralization  with 
sodium  carbonate,  in  order  to  remove  the  sodium  chloride.  At  the 
end  of  this  time,  the  globulin  solution  remaining  in  the  dialyzer  is  fil- 

^Hiss  and  Atkinson,  Jour.  Exper.  Med.,  v,  1900. 

2  Gibson,  Jour,  of  Biol.  Chem.,  i,  1906. 

3  Dr.  Banzhaf,  personal  communication. 

» Gibson  and  Collins,  Jour,  of  Biol.  Chem.,  iii,  1907. 


220  INFECTION  AND   IMMUNITY 

tered  through  a  Berkefeld  candle  for  the  purpose  of  sterilization,  after 
the  addition  of  0.8  per  cent  sodium  chlorid.  According  to  Gibson,  this 
method  produces  a  yield  of  antitoxin  which  equals  about  four-fifths 
of  the  original  quantity  but  is  concentrated  five-  to  seven-^fold.  The 
method  has  more  recently  been  modified  as  follows: 

After  heating  to  56°  C,  as  above,  and  cooling,  ammonium  sulphate  is 
added  to  the  serum  to  thirty  per  cent  saturation.  This  brings  down  all 
the  euglobulins.  This  is  then  filtered  and  the  filtrate,  which  contains 
the  pseudoglobulins  with  the  antitoxin,  is  again  precipitated  with 
ammonium  sulphate  in  a  concentration  of  fifty-four  per  cent  of  satura- 
tion. The  precipitate  is  then  separated  on  a  paper,  pressed  to  dryness, 
and  directly  dialyzed.^ 

Park  and  Thorne^  have  found  that  the  use  of  such  concentrated 
antitoxin  is,  therapeutically,  equally  efficient  as  the  unconcentrated, 
and  possesses  the  advantage  of  less  frequently  giving  rise  to  the  sec- 
ondary reactions  in  skin  and  mucous  membranes  occasionally  noticed 
after  the  use  of  ordinary  antitoxin,  and  referable,  probably,  to  some 
other  constituent  of  the  horse  serum. 

Diphtheria  antitoxin  is  therapeutically  used  in  doses  ranging  from 
3,000  to  20,000  units.  For  prophylactic  immunization  of  healthy 
individuals,  about  500  units  should  be  used. 

TETANUS   ANTITOXIN 

Production  of  Tetanus  Antitoxin. — The  production  of  tetanus  anti- 
toxin is,  in  every  way,  analogous  to  that  of  diphtheria  antitoxin.  It 
is  necessary  in  the  first  place  to  produce  a  powerful  tetanus  toxin.  The 
methods  of  procuring  this  will  be  discussed  in  the  section  upon  tet- 
anus toxin,  page  458.  Suffice  it  to  say  here  that  the  most  satisfactory 
method  of  obtaining  toxins  consists  in  cultivating  the  bacilli  upon  veal 
broth  containing  five-tenths  per  cent  to  two  per  cent  sodium  chlorid 
and  one  per  cent  pep  ton.  It  has  been  advised,  also,  that  the  broth  should 
be  neutralized  by  means  of  magnesium  carbonate  rather  than  with 
sodium  hydrate.  The  bacilli  are  cultivated  for  eight  to  ten  days  at  incu- 
bator temperature  and  the  broth  filtered  rapidly  through  Berkefeld 
filters.  The  toxin  may  be  preserved  in  the  hquid  form  with  the  ad- 
dition of  five-tenths  per  cent  carbolic  acid,  or  may  be  preserved  in  the 
dry  state  after  precipitation  with  ammonium  sulphate. 

1  Dr.  Banzhaj,  personal  communication. 

2  Park  and  Throne,  Amer.  Jour.  Med.  Sci.,  Nov..  1906 


PRODUCTION  AND  TESTING   OF  ANTITOXINS  221 

It  is  necessary  to  determine  the  strength  of  the  poison.  This  is  done 
according  to  Behring  ^  by  determining  the  smallest  amount  of  toxin 
which  will  kill  a  white  mouse  of  twenty  grams  weight  within  four  days. 
This  is  most  easily  done  by  making  dilutions  of  the  toxin  ranging  from 
1  :  100  to  1  : 1,000,  and  then  injecting  quantities  of  0.1  c.c.  of  each  of 
these  dilutions  subcutaneously  into  white  mice.  In  this  way,  the  mini- 
mal lethal  dose  is  ascertained.  * 

For  the  actual  production  of  antitoxin,  horses  have  been  generally 
found  to  be  the  most  favorable  animals.  The  horses  should  be  healthy 
and  from  five  to  seven  years  old.  The  first  injection  of  toxin  admin- 
istered to  these  animals  should  be  attenuated  in  some  way.  Vari- 
ous methods  for  accomplishing  this  have  been  in  use.  In  America, 
the  first  injection  of  about  ten  to  twenty  thousand  minimal  lethal  doses  ^ 
(for  mice  of  twenty  grams  weight)  is  usually  made  subcutaneously  to- 
gether wdth  sufficient  antitoxin  to  neutralize  this  quantity.  In  Germany, 
V.  Behring  uses,  for  his  first  injection,  a  much  larger  dose  of  toxin  to 
which  about  0.25  per  cent  of  terchlorid  of  iodin  has  been  added. 
Immediately  after  an  injection,  the  animals  will  usually  show  a  reac- 
tion expressed  by  a  rise  of  temperature,  refusal  of  food,  and  some- 
times muscular  twitching.  A  second  injection  should  never  be  given 
until  all  such  symptoms  have  completely  subsided.  This  being  the  case, 
after  five  to  eight  days  double  the  original  dose  is  given  together  with 
a  neutralizing  amount  of  antitoxin  or  with  the  addition  of  terchlorid  of 
iodin.  Again  after  five  to  eight  days,  a  larger  dose  is  given  and  there- 
after, at  similar  intervals,  the  quantity  of  toxin  is  rapidly  increased.  In 
America  the  neutralizing  antitoxin  is  omitted  after  the  third  or  fourth 
injection;  in  v.  Behring's  laboratory  the  quantity  of  terchlorid  of  iodin 
is  gradually  diminished.  The  increase  of  dosage  is  often  controlled  by 
the  determination  of  the  antitoxin  contents  of  the  animal's  blood  serum. 
The  immunization  is  increased  until  enormous  doses  (500  c.c.)  of  a 
toxin  in  which  the  minimal  lethal  dose  for  mice  is  represented  by 
0.0001  c.c,  or  less,  is  borne  by  the  horse  without  apparent  harm. 

The  antitoxic  serum  is  then  obtained  by  bleeding  from  the  jugular 
vein,  as  in  the  case  of  diphtheria  antitoxin.  It  may  be  preserved  in  the 
liquid  state  by  the  addition  of  five-tenths  per  cent  of  carbolic  acid  or 
four-tenths  per  cent  of  tricresol. 

1  V.  Behring,  Zeit.  f.  Hyg.,  xii,  1892  ;  Deut.  med.  Woch.,  1900. 

2  According  to  Park  the  "horses  receive  5  c.c.  as  the  initial  dose  of  a  toxin 
of  which  1  c.c.  kills  250,000  grams  of  guinea-pig,  and  along  with  this  a  sufficient 
amount  of  antitoxin  to  neutralize  it." 


22.2  INFECTION  AND  IMMUNITY 

Standardization. — The  universal  prophylactic  use  of  tetanus  antitoxin 
has,  as  in  the  case  of  diphtheria  antitoxin,  necessitated  its  standardiza- 
tion. A  variety  of  methods  are  in  use  in  different  parts  of  the  world. 
In  the  following  description  the  American  method  only  will  be  consid- 
ered as  laid  down  under  the  law  of  July,  1908,  and  based  upon  the  work 
of  Rosenau  and  Anderson  ^  at  the  United  States  Hygienic  Laboratories 
at  Washington. 

In  conjunction  with  a  committee  of  the  Society  of  American  Bac- 
teriologists, these  authors  have  defined  the  unit  of  tetanus  antitoxin  as 
follows : 

The  unit  shall  be  ten  times  the  least  amount  of  serum  necessary  to 
save  the  life  of  a  350  gram  guinea-pig  for  ninety-six  hours  against  the 
official  test  dose  of  standard  toxin.  The  test  dose  consists  of  100  minimal 
lethal  doses  of  a  precipitated  toxin  preserved  under  special  conditions 
at  the  hygienic  laboratory  of  the  Public  Health  and  Marine  Hospital 
Service.  (The  minimal  lethal  dose  is  in  this  case,  unlike  Behring's 
minimal  lethal  dose,  measured  not  against  20  gram  mice,  but  against 
350  gram  guinea-pigs.) 

In  the  actual  standardization  of  tetanus  antitoxin,  as  in  that  of  diph- 
theria antitoxin,  the  L^  dose  of  toxin  is  employed.  The  L+  dose  is, 
however,  in  this  case,  defined  as  the  smallest  quantity  of  tetanus  toxin 
that  will  neutralize  one-tenth  of  an  immunity  unit,  plus  a  quantity  of 
toxin  sufficient  to  kill  a  350  gram  guinea-pig  in  jiist  four  days.  At  the 
Hygienic  Laboratory  at  Washington,  a  standard  toxin  and  antitoxin 
are  preserved  under  special  conditions,  and  standard  toxin  and  anti- 
toxin, arbitrary  in  their  first  establishment,  are  kept  constant  by  being 
measured  against  each  other  from  time  to  time.  In  measuring  the  anti- 
toxic serum  thus  preserved,  at  the  Hygienic  Laboratory,  a  mixture  of 
one-tenth  of  a  unit  of  antitoxin  and  100  minimal  lethal  doses  of  the 
standard  toxin  must  contain  just  enough  free  poison  to  kill  the  guinea- 
pig  in  four  days.  This  L^_  dose  of  the  standard  toxin  is  given  out  to 
those  interested  commercially  or  otherwise  in  the  production  of  antitoxin. 

In  measuring  an  unknown  antitoxic  serum  against  this  L..^.  dose  of 
toxin,  a  large  number  of  mixtures  are  made,  each  containing  the 
L+  dose  of  the  toxin  and  varying  quantities  of  the  antitoxin.  Dilu- 
tions must  always  be  made  with  0.85  per  cent  salt  solution  and  the 
total  quantity  injected  into  the  animals  should  always  be  brought  up  to 


:    1  Rosenau  and  Anderson,  Pub.  Health  and  Mar.  Hosp.  Serv.  U.  S.,  Hyg.  Lab.  Bull. 
43.  IQOS  ,    ■   : 


PRODUCTION  AND  TESTING  OF  ANTITOXINS 


223 


4  c.c.  with  salt  solution  in  order  to  equalize  the  conditions  of  concen- 
tration and  pressure.  The  mixtures  are  then  kept  for  one  hour  at 
room  temperature  in  diffused  light.  After  this  they  are  subcutaneously 
injected  into  a  series  of  guinea-pigs  weighing  from  300  to  400  grams. 
The  following  example  of  a  test  is  taken  from  the  article  by  Rosen?  u 
and  Anderson  quoted  above. 


No.  of 
Guinea- 

Weight  of 

Guinea-pig 

(Grams.) 

Subcutaneous  Injection  of  a 
Mixture  op 

Time  of  Death. 

pig. 

Toxin 
(Test  Dose). 

Antitoxin. 

1 

2 

3 

4 

5 

360 
350 
350 
360 
350 

Gram. 

0.0006 
.0006 
.0006 
.0006 
.0006 

c.c. 

0.001 
.0015 
.002 
.0025 
.003 

2  days  4  hours 
4  days  1  hour 
Symptoms 
Slight  symptoms 
No  symptoms 

In  this  series  the  guinea-pig,  receiving  0.0015  c.c,  of  the  antitoxin,  died 
in  approximately  four  days;  0.0015  c.c.  therefore  represents  one-tenth 
of  an  immunity  unit. 

In  therapeutically  employing  antitoxin  for  prophylactic  purposes, 
about  1,500  units  should  be  employed. 


CHAPTER  XV 
LYSINS,  AGGLUTININS,   PRECIPITINS,   AND  OTHER  ANTIBODIES 

LYSINS 

In  the  immediately  preceding  sections,  we  have  dealt  solely  with 
immunity  as  it  occurs  where  soluble  toxins  play  an  important  part 
and  in  which  antitoxins  are  developed  in  the  immunized  subject.  There 
are  many  species  of  pathogenic  bacteria,  however,  which  stimulate 
the  production  of  little  or  no  antitoxic  substance  when  introduced  into 
animals,  and  the  resistance  of  the  immunized  animal  can  not,  therefore, 
be  explained  by  the  presence  of  antitoxin  in  the  blood. 

V.  Fodor,^  Nuttall,^  Buchner,^  and  others  had  in  1886  and  the  years 
following  carried  on  investigations  which  showed  that  normal  blood 
serum  possessed  the  power  of  killing  certain  of  the  pathogenic  bacteria. 
Nuttall,  working  under  the  direction  of  Fliigge,  made  the  important  dis- 
covery that  this  bactericidal  power  became  gradually  diminished  with 
time,  and  could  be  experimentally  destroyed  by  exposure  of  the  serum 
to  a  temperature  of  56°  C.  for  one-half  hour.  Buchner,  who  confirmed 
and  extended  the  observations  of  Nuttall,  called  this  thermolabile  sub- 
stance upon  which  the  bactericidal  character  of  the  serum  seemed  to 
depend  "alexin.'' 

Our  knowledge  of  the  bactericidal  action  of  serum  was,  soon  there- 
after, extensively  increased  by  the  discovery,  by  Pfeiffer  and  Isaeff,* 
that  cholera  spirilla  injected  into  the  peritoneal  cavity  of  a  cholera- 
immune  guinea-pig  were  promptly  killed  and  almost  completely  dis- 
solved. The  same  phenomenon  could  be  observed  when  the  spirilla, 
mixed  with  fresh  immune  serum,  were  injected  into  the  peritoneum  of  a 
normal  guinea-pig. 

The  processes  observed  by  Pfeiffer  as  taking  place  intraperitoneally 
were  soon  shown  by  Metchnikoff,^  Bordet,®  and  others  to  take  place, 
though  to  a  lesser  extent,  in  vitro.    Bordet,  furthermore,  observed  that 

1 V.  Fodor,  Deut.  med.  Woch.,  1886.  2  Nuttall,  Zeit.  f.  Hyg.,  1886. 

»  Buchner,  Cent,  f .  Bakt. ,  1889.  *  Pfeiffer  und Isaeff,  Zeit.  f .  Hyg.,  1894. 

»  Metchnikoff,  Ann.  de  Tinst.  Pasteur,  1895.  »  Bordet,  ibid.;,  1S95. 

224 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  225 

the  bacteriolytic  digestive  power  of  such  immune  serum,  when  destroyed 
by  heating,  or  after  being  attenuated  by  time,  could  be  restored  by  the 
addition  to  it  of  small  quantities  of  normal  blood  serum.  It  could,  in 
other  words,  be  "reactivated"  by  normal  serum.  From  this  obser- 
vation Bordet  drew  the  conclusion  that  the  bactericidal  or  bacteriolytic 
action  of  the  serum  depended  upon  two  distinct  substances.  The  one 
present  in  normal  serum  and  thermolabile,  he  conceived  to  be  identical 
with  Buchner's  alexin.  The  other,  more  stable,  produced  or  at  least 
increased  in  the  serum  by  the  process  of  immunization,  he  called  the 
"sensitizing  substance."  This  substance,  he  believed,  acting  upon 
the  bacterial  cells,  rendered  them  vulnerable  to  the  action  of.  the  alexin. 
Without  the  previous  preparatory  action  of  the  "sensitizing  substance" 
the  alexin  was  unable  to  act.  Without  the  co-operation  of  alexin,  the 
"sensitizing  substance  "  produced  no  visible  effects. 

Bordet's  interpretation  of  the  phenomenon  of  lysis  differs  essentially 
from  that  of  Ehrlich,  in  that  both  active  serum  components  are  con- 
ceived by  him,  though  independent,  to  act  directly  upon  the  bacterial 
cell.  A  few  years  later,  Bordet  was  able  to  show  that  exactly  analogous 
conditions  governed  the  phenomenon  known  as  "hemolysis"  or  dis- 
integration of  red  blood  cells. 

It  had  been  known  for  many  years  that  in  the  transfusion  of  blood 
from  an  animal  of  one  species  into  an  animal  of  another  species,  in- 
jury was  done  to  the  red  corpuscles  which  were  introduced.  Observed 
in  the  test  tube,  the  red  cells  in  the  heterologous  serum  were  seen  to 
give  up  their  hemoglobin  in  the  fluid,  the  mixture  taking  on  the  red 
transparency  characteristic  of  what  is  known  as  "laked"  blood.  Buch- 
ner,^  in  his  alexin  studies,  had  shown  that  the  blood-cell  destroying 
action  of  the  normal  serum  was  subject  to  the  same  laws  as  the  bac- 
tericidal power  of  similar  serum,  in' that  it  was  destroyed  by  heating, 
and  he  assumed  that  both  the  bacteriolytic  and  the  hemolytic  action 
of  normal  serum  were  due  to  the  same  "  alexin. '^  Metchnikoff ,^  more- 
over, had  pointed  out  the  striking  analogy  between  the  two  phenomena 
as  early  as  1889. 

Bordet  ^  now  observed  that  the  blood  serum  of  guinea-pigs  previously 
treated  with  the  defibrinated  blood  of  rabbits  developed  marked  powers 
of  dissolving  rabbits^  corpuscles,  and  that  this  hemolytic  action  could 


»  BiLchner,  Arch,  f,  Hyg,,  xvii,  1893;  Waremberg,  Arch.  d.  m^.  exper.,  1891. 
2  Metchnikoff,  Ann.  de  Tinst.  Pasteur,  1889. 
8  Bordet,  Ann.  4e  I'inet.  Pasteur,  t,  xii, 


226  INFECTION  AND  IMMUNITY 

be  destroyed  by  heating  to  56°  C,  but  "reactivated"  by  the  addition  of 
fresh  normal  serum.  He  had  thus  produced  an  immune  hemolysin, 
just  as  Pfeiffer  had  produced  immune  bacteriolysin,  and  had  demon- 
strated the  complete  parallelism  which  existed  between  the  two  phe- 
nomena. 

A  practical  test-tube  method  was  thus  given  for  the  investigation  of 
the  lysins,  just  as  a  practical  test-tube  method  for  antitoxin  researches 
had  been  developed  by  Ehrlich  in  his  ricin-antiricin  experiments. 

The  path  of  investigation  thus  pointed  out  by  Bordet  was  soon  ex- 
plored in  greater  detail  by  EhrKch  and  Morgenroth.^  The  reasoning 
which  Ehrlich  had  applied  in  explaining  the  production  of  antitoxins 
was  thought,  by  these  observers,  to  be  equally  applicable  to  the  phe- 
nomena of  bacteriolysis  and  hemolysis. 

Since  the  thermolabile  substance  or  alexin,  renamed  by  Ehrlich 
^'  complement,"  was  already  present  in  normal  serum  and  had  been  shown 
to  be  little,  if  at  all,  increased  during  the  process  of  immunization, 
this  substance  could  have  but  little  relation  to  the  changes  taking  place 
in  the  animal  body  as  immunity  was  acquired.  The  more  stable  serum- 
component,  however,  the  "substance  sensibilisatrice "  of  Bordet,  or, 
as  Ehrlich  now  called  it,  the  "immune  body,''  was  the  one  which  seemed 
specifically  called  forth  by  the  process  of  active  immunization.  EhrHch 
argued,  therefore,  that  when  bacteria  or  blood  cells  were  injected  into 
the  animal,  certain  atom-groups  or  chemical  components  of  the  injected 
substances  were  united  to  other  atom-groups  or  "  side  chains "  of  the 
protoplasm  of  the  tissue  cells.  These  "  side  chains  "  or  receptors,  then 
reproduced  in  excess  and  finally  thrown  free  into  the  circulation,  con- 
stituted the  "immune  body."  The  immune  body,  therefore,  he  con- 
cluded, must  possess  atom  complexes  which  endow  it  with  specific 
chemical  affinity  for  the  bacteria  or  red  blood  cells  used  in  its  produc- 
tion. This  contention  was  supported  by  Ehrlich  and  Morgenroth  by 
an  ingenious  series  of  experiments. 

Having  in  their  possession,  at  that  time,  the  blood  serum  of  a  goat 
immunized  against  the  red  blood  cells  of  a  sheep,  they  inactivated  it 
(destroyed  the  complement  or  alexin)  by  heating  to  56°  C.  The  serum 
then  contained  only  the  "substance  sensibilisatrice "  or  immune  body. 
To  this  inactivated  serum  they  added  sheep's  red  corpuscles,  without 
obtaining  hemolysis.  Having  left  the  inactive  serum  and  the  sheep's 
corpuscles  in  contact  with  each  other  for  some  time,  they  separated 

1  Ehrlich  und  Morgenroth,  Bed.  klin,  Wogh-j,  1,  X899. 


LYSINS,  AGGLUTININS,   PRECIPITINS,  ETC.  227 

them  by  centrifugalization.  To  the  supernatant  fluid,  they  now  added 
sheep-blood  corpuscles  and  normal  goat  serum  (complement)  and  found 
that  no  hemolysis  took  place.  The  immune  body  had  apparently  gone 
out  of  the  serum.  The  red  cells  which  had  been  in  contact  with  the  serum 
and  separated  by  the  centrifuge  were  then  washed  in  salt  solution  and 
to  them  complement  was  added  in  the  form  of  fresh  normal  serum. 


Coropletneni^ 


BodyCell 

Fig.   57. — Ehrlich's  Conception  of  Cell-Receptors,  Giving  Risb   to  Lytic 
Immune  Bodies  (Haptines.  of  the  Third  Order). 

Hemolysis  occurred.  It  was  plain,  therefore,  that  the  immune  body 
of  the  inactivated  serum  had  gone  out  of  solution  and  had  become  at- 
tached to  the  red  blood  cells,  or,  as  Ehrlich  expressed  it,  the  immune 
body  by  means  of  its  '' haptophore "  atom-group  had  become  united 
to  the  corpuscles.    In  contrast  to  this,  if  normal  goat  serum  (containing 


Complement 


Cell  used  for  immunijln^ 

Fig.    58. — Complement,    Amboceptor    or  Immune    Body,    and    Antigen    Orv 
Immunizing  Substance. 

complement  only)  was  added  to  sheep  corpuscles  and  separated  again 
by  centrifugalization,  the  supernatant  fluid  was  found  to  be  still  capable 
of  reactivating  inactivated  serum  (immune  body) .  This  he  interpreted 
as  proving  that  the  complement  was  not  bound  to  the  corpuscles  directly.' 
If  the  three  factors  concerned — corpuscles,  immune  body,  and 
complement — were  mixed  and  the  mixture  kept  at  0°  C,  no  hemolysis 


228  INFECTION  AND  IMMUNITY 

occurred;  yet,  centrifugalized  at  this  temperature,  immune  body  wag 
found  to  have  become  bound  to  the  corpuscles,  the  complement  re- 
maining free  in  the  supernatant  fluid.  If  the  same  mixture,  however, 
was  exposed  to  37°  C,  hemolysis  promptly  occurred. 

From  this,  Ehrlich  concluded  that  complement  did  not  directly 
combine  with  the  corpuscles,  but  did  so  through  the  intervention  of 
the  immune  body.  This  immune  body,  he  reasoned,  possessed  two  die 
tinct  atom-groups  or  haptophores;  one,  the  cytophile  haptophore  group, 
with  strong  affinity  for  the  red  blood  cell;  the  other,  or  complemento- 
phile  haptophore  group,  with  weaker  avidity  for  the  complement.  Be- 
cause of  this  double  combining  power,  Ehrlich  speaks  of  the  immune 
body  as  "amboceptor."  These  views  are  graphically  represented  in 
Figs.  57  and  58. 

Thus,  according  to  Ehrlich  and  his  pupils  the  alexin,  or  comple- 
ment, acts  upon  the  antigen  indirectly  only  through  the  "zwischen- 
koerper"  or  "amboceptor."  Bordet^  claims,  however,  and  in  our 
opinion  rightly  so,  that  this  conception  is  not  justified  by  observed  facts. 
The  only  thing  that  we  actually  know  is  that  alexin  or  complement 
does  not  go  into  union  with  the  unsensitized  antigen,  but  that  the  ac- 
tivity of  the  alexin  upon  the  antigen  is  made  possible  only  by  prelim- 
inary sensitization  or  by  union  with  the  specific  antibody.  The  specific 
antibody  sensitizes  to  the  action  of  the  complement  but  is  not  neces- 
sarily a  sort  or  bridge  with  an  antigenophile  group  and  a  complemento- 
phile  group.  The  difference  is  a  fundamental  one,  and  it  would  seem 
to  us  that  Bordet's  view  is  more  conservative.  For  this  reason  it  seems 
better  to  refer  to  the  substances  involved,  alexin. or  complement,  as 
sensitizer  instead  of  amboceptor,  since  sensitizer  expresses  only  exactly 
what  happens,  whereas  amboceptor  implies  a  theory,  that  is,  inter- 
mediation of  this  substance  between  complement  and  antigen. 

AGGLUTININS 
Although  Metchnikoff  ^  ^nd  Charrin  and  Roger  ^  had  noticed  pecul- 
iarities in  the  growth  of  bacteria  when  cultivated  in  immune  sera,  which 
were  unquestionably  due  to  agglutination,  the  first  recognition  of  the 
agglutination  reaction  as  a  separate  function  of  immune  sera  was  the 
achievement  of  Gruber  and  Durham.  While  investigating  the  Pfeiffer 
reaction  with  B.  coli  and  the  cholera  vibrio,  Gruber  and  Durham  * 

^Bordet,  A  Resume  of  Immunity  in  ''Studies  in  Immunity."    Transl.  by  Gay 
'Wiley  &  Son,  1909.  2  Metchnikoff,  "Etudes  sur  Timmunite,"  IV  Memoir,  1891 

'  Charrin  et  Roger,  Compt.  rend,  de  la  soc.  de  biol.,  1889. 
4  Gruber  und  Durham^  Munch,  med.  Woch.,  1896. 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  229 

noticed  that  if  the  respective  immune  sera  were  added  to  bouillon  cul- 
tures of  these  two  species,  the  cultures  would  lose  their  turbidity  and 
flake-like  clumps  would  sink  to  the  bottom  of  the  tube,  the  supernatant 
fluid  becoming  clear.  Gruber,  at  the  same  time,  called  attention  to  .the 
fact  that  immune  sera  would  affect  in  this  way  not  only  the  microor- 
ganism used  in  their  production,  but,  to  a  less  energetic  extent,  other 
closely  related  bacteria  as  well. 

Widal,  very  soon  after  Gruber  and  Durham's  announcement,  ap- 
plied the  agglutination  reaction  to  the  practical  diagnosis  of  typhoid 
fever,  finding  that  the  serum  of  patients  afflicted  with  this  disease 
showed  agglutinating  power  over  the  typhoid  bacillus  at  early  stages 
in  the  course  of  the  fever.  The  reaction,  thus  practically  applied  to 
clinical  diagnosis,  was  soon  shown  to  be  of  great  importance  in  its 


Fig.  59. — Microscopic  Agglutination  Reaction. 

bearing  on  bacteriological  species  differentiation.  Since  animals  .im- 
munized against  a  definite  species  of  bacteria  acquire  in  their  s^ra 
specific  agglutinating  powers  for  these  bacteria  and  at  best  only  slight 
agglutinating  powers  for  other  species,  immune  sera  can  be  used  ex- 
tensively in  differentiating  between  bacterial  varieties. 

Agglutination  may  be  observed  microscopically  or  macroscopically. 
Bacteria  brought  into  contact  with  agglutinating  serum  in  the 
hanging  drop  rapidly  lose  their  motility,  if  motile,  as  in  the  case  of 
typhoid  bacilli,  and  gather  together  in  small  clumps  or  masses.  The 
microscopic  picture  is  striking  and  easily  recognized  and  the  reaction 
takes  place  with  varying  speed  and  completeness,  according  to  the 
strength  of  the  agglutinating  serum. 

As  the  reaction  approaches  completeness,  tha  clumps  grow  larger^, 
16 


230 


INFECTION  AND  IMMUNITY 


individual  microorganisms  become  more  and  more  scarce,  finally 
leaving  the  medium  between  clumps  entirely  clear.  While  the  clumping 
of  a  motile  organism  suggests  that  motility  has  something  to  do  with  the 
coming  together  in  clumps,  it  nevertheless  has  no  relation  whatever 
to  agglutination,  motile  and  non-motile  organisms  alike  being  subject 
to  the  reaction. 

Macroscopically  observed,  in  small  test  tubes  or  capillary  tubes,  ag- 
glutination evidences  itself  by  the  formation  of  flake-like  masses  which 


Fig.  60. — Macroscopic  Agglutination.  Dilutions  from  1  in  10  to  1  in  1,000. 
The  first  tube  contains  a  1  :  20  control  with  the  bacteria  and  normal  serum. 
Agglutination  complete  in  the  tubes  marked  10,  20,  50,  100. 

settle  into  irregular  heaps  at  the  bottom,  leaving  the  supernatant 
fluid  clear,  in  distinct  contrast  to  the  even  flat  sediment  and  the  clouded 
supernatant  fluid  of  the  control.  Macroscopically,  too,  agglutination 
is  evidenced  when  bacteria  are  grown  in  broth  to  which  immune  serum 
has  been  added.  Instead  of  evenly  clouding  the  broth,  the  micro- 
organisms develop  in  clumps  or  chains. 

Another  phenomenon  probably  produced  by  agglutinins  is  the  so- 


LYSINS,  AGGLtJTINlNS,  PRECIPITIiSTS,  ETC.  231 

called  "thread-reaction''  of  Pfaundler.^  This  consists  in  the  forma- 
tion of  long  convoluted  threads  of  bacterial  growth  in  the  hanging  drop 
of  dilute  immune  serum  after  twenty-four  hours.  Very  strict  specificity 
is  attributed  to  this  reaction  by  Pfaundler. 

Agglutinins  act  upon  dead  as  well  as  upon  living  bacteria.  For  the 
microscopic  tests  bacterial  emulsions  killed  by  formalin  were  intro- 
duced by  Neisser. 

Ficker  ^  has  recently  succeeded  in  preparing  an  emulsion  of  typhoid 
baciUi,  which  is  permajient  and  may  be  kept  indefinitely,  and  may  be 
employed  for  macroscopic  agglutinations.^ 

Attention  has  been  called  by  various  workers  to  a  source  of  error  in 
all  these  methods,  known  as  pseudo-climaping.'*  The  causes  for  such 
clumping  not  due  to  agglutinins  seem  to  lie  in  the  presence  of  blood  cells 
in  the  serimi  or  excessive  acidity  of  the  culture  medium.^  In  fact,  ag- 
glutination of  bacteria  by  acids  in  definite  concentration  can  be  carried 
out  and  seems  to  depend  directly  upon  the  hydrogen  ion  concentration. 

While  the  microscopic  methods  are  more  suitable  for  clinical-diag- 
nostic purposes,  because  of  the  smaller  amounts  of  blood  required,  the 
macroscopic  tests  are  far  preferable  for  the  purposes  of  bacterial  differen- 
tiation and  research.  Greater  exactitude  of  dilution  is  possible  when 
dealing  with  larger  quantities;  microscopic  unevenness  in  the  bacterial 
emulsion  does  not  become  a  source  of  error;  and  positive  and  negative 
reactions  are  more  sharply  defined. 

Nature  of  Agglutinins. — Gruber  and  Durham,^  the  discoverers  of  ag- 
glutinins, at  first  advanced  the  opinion  that  the  agglutinins  were  iden- 
tical with  the  immime  body  concerned  in  the  Pf  eiffer  reaction,  which  by 
injuring  the  bacteria  rendered  them  susceptible  to  the  alexins.  Pfeiffer  ^ 
and  Kolle  ^  soon  showed,  however,  that  by  the  addition  of  cholera  vibrio 
to  immune  serum,  the  agglutinins  could  be  completely  absorbed,  or  used 
up,  while  bacteriolytic  substances  still  remained.  The  same  authors 
demonstrated  that  immune  serum,  preserved  for  several  months,  would 
lose  its  agglutinins  without  a  corresponding  loss  of  bacteriolytic  power. 
It  has  been  variously  shown  since  then,  by  these  and  other  authors, 
that  the  agglutinins  and  the  bactericidal  substances  are  in  no  way  parallel 

1  Pfaundler,  Cent.  f.  Bakt.,  xxiii,  1898.        2  picker,  Berl.  klin.  Woch.,  1903. 

'  Exact  method  of  production  of  "Picker's  Diagnosticum"  is  a  proprietary  secret. 

^Savage,  Jour,  of  Path,  and  Bact.,  1901. 

5  Biggs  and  Park,  Amer.  Jour,  of  Med.  Sci.,  1897;  Bloch^  Brit.  Med.  Jour.,  1897. 

«  Loc.  cit.  7  Pfeiffer,  Deut.  med.  Woch.,  1896. 

»  Pfeiffer  und  KoUe,  Cent,  f .  Bakt.,  xx,  1896. 


232  INFECTION  AND  IMMUNITY 

in  their  development,  and  that  strongly  agglutinating  sera  may  be  ex- 
tremely weak  in  bactericidal  substances  and  vice  versa.  We  ourselves 
are  not  at  all  sure  that  this  proves  sufficiently  that  agglutinins  and 
bacteriolysins  are  distinct  substances.  Whether  or  not  agglutinins 
possess  any  direct  protective  function  can  not  at  present  be  stated 
with  certainty.  Metchnikoff  ^  assigns  to  them  a  purely  secondary  role.. 
As  a  matter  of  fact,  agglutinated  bacteria  ^  are  not  killed  by  the  act 
of  agglutination  and  are  often  as  virulent  as  non-agglutinated  cultures. 
Recent  work  by  Bull  seems  to  indicate  that  bacteria  are  agglutinated 
in  vivo  as  a  sort  of  preliminary  step  to  phagocytosis. 

The  agglutinins,  furthermore,  unlike  the  bactericidal  substances  in 
sera,  remain  active  after  exposure  to  temperatures  of  over  55°  C,  some 
of  them  withstanding  even  65°  to  70°,  and  can  not  be  reactivated  by  the 
subsequent  addition  of  normal  serum.  These  facts  definitely  preclude 
the  participation  in  the  reaction  of  the  alexin  or  complement  and  have 
an  important  bearing  upon  Ehrlich's  views  of  their  structiu-e  ^  (p.  238). 

As  a  result  of  these  and  a  multitude  of  other  studies,  the  agglu- 
tinins have  come  to  be  regarded  as  separate  antibodies,  closely  related 
to  the  precipitins. 

The  agglutinins  may  be  chemically  precipitated  out  of  serum  to- 
gether with  the  globulins.  They  do  not  dialyze.  Bordet^  made  the 
observation  that  agglutinins  do  not  act  in  the  absence  of  NaCl.  Whether 
the  presence  of  the  salt  aids  the  reaction  in  a  chemical  or  purely  physical 
way,'  as  Bordet  supposed,  is  uncertain. 

Production  of  Agglutinins. — Just  as  normal  sera  contain  small  quan- 
tities of  bactericidal  substances,  so  do  they  contain  agglutinins  in  small 
amount.  In  a  general  way  these  "normal  agglutinins"  have  the  same 
nature  as  the  immune  agglutinins,  and  their  presence  is  probably  trace- 
able to  the  various  microorganisms  parasitic  upon  the  human  and^ 
animal  body. 

As  a  matter  of  fact,  the  blood  serum  of  new-born  guinea-pigs  hardly 
ever  contains  agglutinin  for  B.  coli,  while  that  of  adults  acts  upon  these 
bacilli  in  dilutions  of  1  :  20.^  Similarly,  infants  show  lower  normal  ag- 
glutinating values  than  adults.  ^ 

1  Metchnikoff,  "LTrnmunite,"  etc.,  1901,  p.  214. 

2  Mesnil,  Ann.  de  Tinst.  Pasteur,  1898= 

» Pane,  Cent.  f.  Bakt.,  1897;  Trumpp,  Arch.  f.  Hyg.,  1898;  Forster,  Zeit.  f.  Hyg., 
xxiv.     ■*  Bordet,  Ann.  de  I'inst.  Pasteur,  1899. 

B  Kraus  und  Low,  Gesell.  d.  Aerzte,  Wien,  1899. 
e  Pfaundler,  Jahrb.  f .  Kinderheilk.,  Bd.-50.    . 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC  233 

Agglutinins  may  be  produced  in  the  sera  of  animals  by  the  intro- 
duction of  microorganisms  subcutaneously,  intravenously,  or  intraperi- 
toneally.  The  intravenous  method  seems  to  give  the  most  abundant  and 
speedy  results.  1  The  formation  of  agglutinins  is  a  reaction  to  the  body- 
stances  of  the  bacteria  themselves,  rather  than  to  their  toxic  prod- 
ucts. Thus  agglutinins  are  produced  in  response  to  the  introduction 
of  dead  bacteria  and  soluble  extracts  of  cultures.  Pathogenicity  ^  does 
not  influence  agglutinin  formation  to  any  great  extent,  non-pathogenic 
as  well  as  pathogenic  giving  rise  to  these  substances  in  serum.  As  a 
rule,  however,  agglutinins  are  more  easily  produced  against  avirulent 
than  against  fully  virulent  strains  of  bacteria  of  the  same  species. 

Agglutinins  can  be  produced  with  all  the  known  bacteria,  but  great 
difficulty  may  be  experienced  in  producing  them  with  capsulated  or- 
ganisms such  as  the  pneumococcus  mucosum  and  the  Friedlander  bac- 
illus, since  the  capsule  seems  to  insulate  such  bacteria  against  reactions 
with  serum.  It  is  possible  to  agglutinate  such  capsulated  bacteria 
often  only  by  the  method  of  Porges,  the  preliminary  destruction  of 
the  capsule  with  weak  acid  and  heat.  As  a  rule,  the  agglutinins  appear 
in  the  blood  of  animals  three  to  six  days  after  the  introduction  of  bac- 
teria. From  the  third  to  the  sixth  day  they  rapidly  increase  to  a  max- 
imum at  the  seventh  to  thirteenth  day.  They  then  fall  off  rapidly 
until  they  reach  a  level  at  which  they  remain  for  a  long  period  without 
very  considerable  change.  Curves  to  illustrate  these  phases  have  been 
constructed  by  Jorgensen  and  Madsen.^ 

The  Reaction  between  Agglutinin  and  Agglutinin-Stimulating  Sub- 
stances {Agglutinogen). — The  fact  that  agglutinin  can  be  removed  from, 
or  absorbed  out  of,  serum  by  the  specific  bacilli  which  have  led  to  its 
formation  indicates  that  there  is  in  the  act  of  agglutination  a  combi- 
nation between  the  agglutinin  and  the  agglutinin-stimulating  substance 
(agglutinogen).  It  is  likely  that  this  combination  is  of  a  chemical  na- 
ture, since,  as  we  have  mentioned,  agglutinins  result  from  the  injection 
of  bacterial  extracts  as  well  as  from  the  introduction  of  living  bacteria. 
The  probability  that  the  process  follows  chemical  laws  of  combination 
is  furthermore  strengthened  by  the  work  of  Joos  ^  and  others,  who 
have  demonstrated  that  definite  quantitative  relations  exist  between 
the  agglutinin-stimulating  substances  and  the  agglutinins.  Every 
agglutination    reaction,    therefore,  will  vary  in    its    degree    of    com- 

1  Hoffmann,  Hyg.  Rundschau,  1903.  ^  j^icolle,  Ann.  de  Tinst.  Pasteur,  1898. 

3  Jorgensen  and  Madsen,  Festschrift,  Kopenhagen,  1902. 
*  Joos,  Zeits.  f .  Hyg.,  xxxvi,  1901. 


234  INFECTION   AND    IMMUNITY 

pletehess  with  the  quantities  of  agglutinin  and  agglutinogen,  a  fact 
which  makes  it  necessary,  especially  for  clinical  tests,  to  preserve  a 
certain  uniformity  in  the  quantity  and  density  of  the  bacterial  culture 
or  emulsion  employed. 

Specificity. — From  the  very  beginning,  Gruber  and  Durham  ^  had 
claimed  specificity  for  the  agglutination  reaction,  and  in  this  sense  it  was 
clinically  utilized  by  Widal  for  the  diagnosis  of  typhoid  fever.  It  was 
noticed,  however,  even  by  these  earliest  workers,  that  the  serum  of  an 
animal  immunized  against  one  microorganism  would  often  agglutinate, 
to  a  less  potent  degree,  other  closely  related  species.  Thus,  the  serum 
of  a  typhoid-immune  animal  may  agglutinate  the  typhoid  bacillus  in 
dilutions  of  1  : 1,000,  and  the  colon  bacillus  in  dilutions  as  high  as  1  :  200; 
while  the  agglutinating  power  of  normal  serum  for  the  colon  bacillus 
rarely  exceeds  1  :  20.  The  specificity  of  the  reaction  for  practical  pui 
poses,  thus,  is  not  destroyed  if  proper  dilution  is  carried  out,  the  degree 
of  agglutinin  formation  being  always  far  higher  for  the  specific  organism 
used  in  immuaifeation  than  it  is  for  allied  organisms.  The  specific 
immune-agglutinin  in  such  experiments  is  spoken  of  as  the  chief  ag- 
glutinin (hauptagglutinin) ,  and  the  agglutinins  formed  parallel  with  it, 
as  the  partial  agglutinin  (metagglutinin) ,  terms  introduced  by  Wasser- 
mann.  Hiss  has  spoken  of  these  as  major  and  minor  agglutinins.  The 
relative  quantities  of  the  specific  chief  agglutinin  and  partial  agglutinins 
present  in  any  immune  serum  depend  upon  the  individual  cultures  used 
for  immunization,  and  the  phenomenon  is  probably  dependent  upon 
the  fact  that  certain  elements  in  the  complicated  bacterial  cell-body 
may  be  common  to  several  species  and  find  common  receptors  in  the 
animal  body.  Whenever  an  immune  serum  agglutinates  a  number  of 
members  of  the  group  related  to  the  specific  organism  used  for  its  produc- 
tion, the  reaction  is  spoken  of  "group  agglutination." 

The  partial  agglutinins  (metagglutinins)  have  been  extensively 
studied  by  Castellani  ^  and  others,  by  a  method  spoken  of  as  the  "  ab- 
sorption method.''  This  consists  in  the  separate  addition  of  bacterial 
emulsions  (agglutinogens)  of  the  various  species  concerned  in  a  group 
agglutination,  to  the  agglutinating  serum.  In  this  way,  specific  and  par- 
tial agglutinins  can  be  separately  removed  from  the  immune  serum  by 
absorption — each  by  its  corresponding  agglutinogen.  In  such  experi- 
ments all  agglutinins  will  be  removed  by  the  organisms  used  for  im- 

1  Gruber  und  Durham,  loc.  cit. 

3  Castellani,  Zeits.  f.  Hyg.,  xl,  1902. 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  235 

munization,  a  partial  removal  only  resulting  from  the  addition  of  allied 
strains.  This  method  has  thrown  much  light  upon  the  intimate  relations 
existing  between  members  of  various  bacterial  species,  and  has  been 
particularly  valuable  in  the  study  of  the  typhoid-colon-dysentery 
group.  It  is  important  to  mention,  however,  that  "groups"  as  de- 
termined by  agglutination  tests  do  not  always  correspond  to  classi- 
fications depending  upon  morphological  and  cultural  characteristics. 

An  interesting  phenomenon  of  great  practical  importance,  which 
has  been  noticed  by  a  number  of  observers,  and  which  may  often  be 
encountered  in  routine  agglutination  tests,  is  the  frequent  failure  of  a 
strongly  agglutinating  serum  to  produce  agglutination  if  used  in  concen- 
tration, while  in  dilutions  it  produces  a  characteristic  reaction.  This  has 
been  explained  theoretically  by  what  is  known  as  the  '^proagglutinoid 
zone.''  It  is  assumed  that  agglutinins  may  deteriorate  as  do  toxins  and 
be  converted  into  substances  which  are  capable  of  combining  with  agglu- 
tinogen without  causing  agglutination.  Such  substances,  as  we  will 
see  in  discussing  Ehrlich's  views  on  the  structure  of  agglutinins,  may 
havd  a  stronger  affinity  for  agglutinogen  than  the  agglutinins  them- 
selves, and  are,  therefore,  termed  "proagglutinoids."  In  strongly 
agglutinating  sera  these  proagglutinoids  may  be  present  in  considerable 
quantities  and  prevent  the  combination  of  agglutinin  with  agglutinogen. 
In  dilution,  this  proagglutinoid  action  would  naturally  become  weaker 
and  of  no  actual  significance  in  obscuring  the  reaction. 

Agglutination,  like  other  immune  phenomena,  is  a  manifestation  of 
broad  biological  laws  and  not  limited  to  bacteria.  Thus,  as  hemolysins 
are  produced  by  the  injection  of  red  blood  cells,  so  hemagglutinins,  or 
substances  which  clump  together  red  blood  cells,  are  similarly  formed. 

The  theoretical  considerations  concerning  the  nature  of  agglutinins 
are  discussed  below,  together  with  a  similar  section  on  the  precipitins. 

PRECIPITINS 

R.  Kraus,^  of  Vienna,  demonstrated  that  the  sera  of  animals  im- 
munized against  B.  pestis,  B.  typhosus,  and  Vibrio  cholerae,  when 
mixed  with  the  clear  filtrate  of  bouillon  cultures  of  the  respective  or- 
ganisms, produce  macroscopically  visible  precipitates.  These  precipi- 
tates occurred  only  when  filtrate  and  immune  serum  were  homologous, 
i.  e.,  when  the  animal  from  which  the  serum  had  been  obtained  had  been 
immunized  by  the  same  species  of  microorganism  as  that  which  was 
used  in  the  test;  it  was  for  this  reason  Kraus  spoke  of  them  as  "specific 

1  Kraus,  Wien.  klin.  Woch.,  1897. 


236  INFECTION  AND  IMMUNITY 

precipitates."  It  was  evident,  therefore,  that  during  the  process  of  active 
immunization  with  these  organisms,  a  specific  antibody  had  been  pro- 
duced in  the  serum  of  the  treated  animal,  which,  because  of  its  precipitat- 
ing quaUty,  was  named  ''precipitin."  This  pecuhar  reaction  was  soon 
found  to  hold  good,  not  only  for  the  bacteria  used  by  Kraus,  but  also  for 
other  bacteria,  few  failing  to  stimulate  the  production  of  specific  precipi- 
tins in  the  sera  of  immunized  animals.  The  phenomenon  of  precipitation, 
however,  is  not  limited  to  bacterial  immunization,  but  has  been  found, 
like  the  phenomena  of  agglutination  and  lysis,  to  depend  upon  biolog- 
ical laws  of  broad  application.  .  Thus,  Bordet  ^  found  that  the  blood 
serum  of  rabbits  treated  with  the  serum  of  the  chicken  gave  a  specific 
precipitate  when  mixed  with  chicken  serum.  Tchistovitch  ^  demon- 
strated a  similar  reaction  with  the  sera  of  rabbits  treated  with  horse  and 
eel  sera.  By  the  injection  of  milk,  Wassermann,^  Schiitze,^  and  others 
produced  an  antibody  which  precipitated  the  casein  of  the  particular 
variety  of  milk  employed  for  immunization.  The  reaction  was  thus 
applicable  to  many  albuminous  substances.  These  substances,  because 
of  their  precipitin-stimulating  quality,  are  called  "precipitinogens." 

Nature  of  Precipitins. — The  precipitins,  hke  the  agglutinins,  may  be 
inactivated  by  heating  to  from  60°  to  70°  C,  and  can  not  be  reactivated 
by  the  addition  of  normal  serum  or  by  any  other  known  method. 
Such  inactivated  precipitin,  however,  while  unable  to  produce  precipi- 
tates, has  not  lost  its  power  of  binding  the  precipitinogen.  This  is 
shown  by  the  fact  that  the  inactivated  precipitin,  when  mixed  with  pre- 
cipitinogen, will  prevent  subsequently  added  fresh  precipitin  from  caus- 
ing a  reaction.  From  these  facts  the  conclusion  has  been  drawn  that 
precipitin,  Hke  toxin,  is  built  up  of  two  atom  groups,^  a  stable  hap- 
tophore  and  a  labile  precipitophore  group.  By  the  destruction  of  the 
latter,  an  inactive,  yet  neutralizing  substance  is  produced  which  is 
spoken  of  as  "precipitoid."  The  precipitoids,  like  protoxoids,  have 
a  higher  affinity  for  precipitinogen  than  the  unchanged  precipitin,  and 
thus  are  able  to  prevent  the  action  of  these. 

Our  own  opinion  would  rather  incline  toward  regarding  the  pre- 
cipitins as  identical  in  structure  with  sensitizer  or  amboceptor — being 
in  fact  "  albuminolysins  "  in  the  sense  of  Gengou.     This  problem  is  too 

1  Bordet,  Ann.  de  I'inst.  Pasteur,  1899. 

2  Tchistovitch,  Ann.  de  I'inst.  Pasteur,  1899. 

3  Wassermann,  Deut.  med.  Woch.,  29,  1900. 
*^c;iMtee,  Zeit.  f.  Hyg.,  1901. 

5  Kram  und  v.  Pirquet,  Cent.  f.  Bakt.,  Orig.  Bd.  xxxii. 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  237 

complex  to  be  discussed  in  detail  in  a  summary  of  immunity  as  brief 
as  the  one  here  presented. 

Specificity. — The  specificity  of  precipitins  is  a  question  of  the 
greatest  importance,  since,  as  we  shall  see,  these  bodies  have  been  used 
extensively  for  the  differentiation  of  animal  proteids.  In  regard  to 
bacterial  precipitins  it  may  be  said  that,  just  as  in  agglutination,  there 
is  in  precipitation  a  certain  degree  of  "group  reaction."  The  pre- 
cipitin obtained  with  a  colon  bacillus,  for  instance,  will  cause  precipita- 
tion with  culture-filtrates  of  closely  allied  organisms,  though  in  a  less 
marked  degree.  According  to  Kraus,  such  confusion  may  be  easily 
overcome  by  the  proper  use  of  dilution  and  quantitative  adjustment, 
similar  to  that  used  in  •agglutination  tests.  Norris  ^  found  that  the 
precipitates  given  by  immune  sera  with  the  filtrates  of  the  homologous 
bacteria  were  invariably  heavier  than  those  given  with  allied  strains 
and  that  the  latter  could  be  eliminated  entirely  by  sufficient  dilution. 

Specificity  becomes  of  still  greater  importance  in  the  forensic  use 
of  the  precipitin  reaction  introduced  by  Uhlenhuth,^  Wassermann  and 
Schtitze,^  and  Stern.*  These  authors  found  that  the  precipitin  reaction 
furnished  a  means  of  distinguishing  the  blood  of  one  species  from  that 
of  another.  Thus,  blood  spots,  dissolved  out  in  normal  salt  solution, 
could  be  recognized  by  this  reaction  as  originating  from  man  or  from 
an  animal,  even  after  months  of  drying  and  in  dilutions  as  high  as  1 :  50,- 
000.  Since  the  value  of  this  test  depends  entirely  upon  the  strict 
specificity  of  the  reaction,  this  question  has  been  studied  with  especial 
care,  notably  by  Nuttall.^  All  who  have  investigated  the  subject  find 
the  only  important  source  of  confusion  in  the  blood  of  the  anthropoid 
apes.  The  specificity  of  the  reaction,  too,  has  been  found  to  depend  very 
closely  upon  the  amount  of  precipitin  in  the  serum  employed.  If  a 
highly  immune  serum  is  insufficiently  diluted,  the  reaction  loses,  much 
of  its  specific  value. ^  This  source  of  error  is  easily  eliminated  in  practice 
by  careful  control  and  titration  of  the  sera  used  for  the  tests. 

Unlike  agglutinins,  precipitins  have,  so  far,  not  been  demonstrated 
in  normal  sera.^ 


1  Norris,  Jour.  Inf.  Dis.,  i,  3,  1904. 

2  Uhlenhidh,  Deut.  med.  Woch.,  xlvi,  1900;  vi  and  xvii,  1901. 

3  Wassermann  und  Schiitze,  Berl.  klin.  Woch.,  vi,  1901. 
*  Stern,  Deut.  med.  Woch.,  1901. 

^Nuttall,  Brit.  Med.  Jour.,  i,  1901;  ii,  1902. 

«  Kister  und  Wolff,  Zeit.  f .  Medizinal-Beamte,  1902. 

^  Kraus,  loc.  cit.,  and  Norris,  loc.  cit. 


238  INFECTION  AND  IMMUNITY 

Theoretical  Considerations  Concerning  Agglutinins   and  Precipitins. — 

We  have  seen  that  Ehrlich  evolved  his  theories  of  antibody  forma- 
tion from  his  early  views  upon  the  absorption  of  nutritive  substances  by 
the  body  cells,  and  we  have  followed,  in  more  or  less  detail,  the  steps  of 
his  reasoning  as  he  developed  his  hypothesis  in  its  application  to  the 
antitoxic  and  the  lytic  substances.  There  still  remained  the  agglutinins 
and  precipitins,  bodies  which  because  of  their  individual  characteris- 
tics can  be  classed  neither  with  the  group  of  antitoxic,  nor  with  that  of 
the  lytic  substances.  These  two  antibodies,  while  by  no  means  identical, 
possess  the  common  characteristics  of  being  more  thermostable  than  the 
bacteriolytic  substances,  and  of  being  insusceptible  to  reactivation  by 
normal  serum.  It  is  plain,  therefore,  that  'both  agglutinating  and 
precipitating  reactions  take  place  without  the  co-operation  of  comple- 
ment.   The  substances  which  give  rise  to  precipitins  and  agglutinins^ 


Cell  Msed  for  ijninumjin^ 

Fig.    61. — Ehrlich's    Conception    of    the    Structure  of  Agglutinins    and 

Precipitins. 

moreover,  are  not  of  the  relatively  simple  soluble  character  of  the  toxins, 
but  are  intrinsic  portions  of  complex  albuminous  molecules,  comparable 
to  and  often  identical  with  the  true  nutritive  substances.  For  these 
reasons  Ehrlich  believes  that  the  cell-receptors  for  the  various  substances 
which  give  rise  to  agglutinins  and  precipitins  are  neither  of  the  simple 
structure  of  the  toxin  receptor,  nor  of  the  double-haptophore  nature  of 
the  bacteriolytic  receptors,  but  contain  a  single  haptophore  group  for 
the  anchorage  of  the  ingested  material  and  at  the  same  time  a  constantly 
attached  zymophore  group  or  ferment  by  means  of  which  the  an- 
chored substance  is  transformed  preparatory  to  its  absorption  by  ihe 
cell  protoplasm.  For  the  sake  of  clearness,  this  form  of  receptor  may 
be  compared  to  a  bacteriolytic  or  hemolytic  amboceptor  with  a  per- 
manently attached  and  inseparable  complement. 

Three  forms  of  receptors,  then,  are  proposed  by  Ehrlich  in  explana- 
tion of  all  known  varieties  of  antibodies.    The  first,  the  simplest  side 


239 


240  INFECTION  AND   IMMUNITY 

chains  of  the  body  cells,  he  calls  "  receptors  or  haptines  of  the  first  order." 
These,  overproduced  and  cast  off,  constitute  the  antitoxin  and  antifer- 
ments.  Next  "  haptines  of  the  second  order  "  are  the  receptors  planned 
both  for  the  anchorage  and  further  digestion  of  antigens.  These,  free 
in  the  circulation,  are  the  precipitins  and  agglutinins.  "Haptines  or 
receptors  of  the  third  order"  are  merely  able  to  anchor  a  suitable  sub- 
stance, but  exert  no  further  action  upon  it  until  re-enforced  by  the  com- 
plement normally  present  in  the  serum.  These,  free  in  the  circulation, 
with  a  chemical  group  having  avidity  for  the  antigen,  and  another 
complementophile  group,  are  the  amboceptors  or  immune  bodies  of 
bacteriolytic,  cytolytic,  and  hemolytic  sera.      (See  Fig.  62.) 

It  is  plain  that  all  these  receptors  while  still  parts  of  their  respec- 
tive cells,  serve  by  their  chemical  affinity  to  attract  and  hold  the  foreign 
substances  injected;  freely  circulating,  on  the  other  hand,  they  serve 
in  preventing  these  substances  from  reaching  the  cells.  As  Behring 
has  aptly  expressed  it,  the  very  elements  which  situated  in  the  animal 
cells  render  the  body  susceptible  to  toxic  substances  serve  to  protect 
when  circulating  freely  in  the  blood. 

Bordet,^  at  present  the  strongest  antagonist  of  Ehrlich's  point  of 
view,  claims  that  the  conception  of  Ehrlich  rests  upon  the  basis  of  a 
number  of  undemonstrated  hypotheses.  He  asserts,  and  with  justice, 
that  it  has  never  been  shown  beyond  question  that  the  antibodies, 
free  in  the  serum,  are  identical  with  the  receptors  of  the  body  cells 
upon  which  the  antigen  originally  acts. 

In  regard  to  agglutinins,  Ehrlich,  as  we  have  seen,  believes  that  it  is 
the  agglutinin  itself  which,  first  uniting  with  its  antigen  by  its  hap- 
tophore  group,  then  causes  clumping  by  its  zymophore  group.  Now, 
as  a  matter  of  fact,  Bordet^  has  shown  that  it  is  not  the  agglutinin  itself 
which  agglutinates,  but  that  agglutinin  with  its  antigen  forms  a  com- 
plex which  is  then  agglutinable  by  the  salt  present  in  the  solution.  This 
conclusion  seems  borne  out  by  the  later  work  of  Gengou,^  Landsteiner 
and  Jagic,^  and  others,  who  have  shown  that  bacteria  which  have  ab- 
sorbed other  substances,  such  as  uranium  compounds,  colloidal  silicic 
acid,  etc.,  are  subsequently  agglutinable  by  salts.  In  consequence, 
from  these  and  other  observations,  Bordet  concludes  that  it  is  neither 
necessary  nor  accurate  for  the  explanation  of  these  phenomena,  to 

'-  Bordet,  Resume  of  Immunity  in  Bordet's  "  Studies  in  Immunity,"  transl.  by  Gay, 
Wiley  &  Sons,  1909. 

2  Bordet,  Ann.  de  I'inst.  Pasteur,  1899.  ^Gengpu,  Annal.  Past.,  1904. 

*  Landsteiner  und  Jagic.  Wien.  klin.  Woch,,  iii,  1904. 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  241 


assume  the  conditions  conceived  by  Ehrlich,  but  that  the  phenomenon 
of  agglutination  consists  primarily  of  the  union  of  the  antibody  with  its 
antigen  in  a  colloidal  suspension,  and  that  the  actual  subsequent  agglu- 
tination is  a  purely  secondary  phenomenon  which  depends  possibly 
upon  a  change  in  the  physical  properties  of  the  emulsion — upon  "its 
colloidal  stability."    A  similar  condition  he  assumes  for  precipitins. 

Without  being  able  in  the  limited  space  available  to  go  into  a  de- 
tailed discussion  of  the  large  volume  of  work  which  has  appeared  on 
this  subject,  we  may  say  that  it  is  our  opinion  at  present  that  the  evi- 
dence largely  points  in  the  direction  indicated  by  Bordet,  namely,  that 
the  essential  feature  of  all  these  reactions  is  the  specific  union  of  an 
antigen  with  its  antibody,  that  thereby  the  physical  or  chemical  con- 
dition of  the  antigen  is  so  changed  that  it  now  becomes  less  stable  and 
is  agglutinated  or  precipitated  by  such  physical  influences  as,  for  in- 
stance, the  presence  of  an  electrolyte.  The  work  of  Neisser  and  Fried- 
mann  ^  has  shown  that  bacteria  that  have  absorbed  agglutinin  are 
agglutinated  by  concentrations  of  salt  far  less  than  is  necessary  to  ag- 
glutinate or  precipitate  the  normal  bacteria. 

Our  own  opinion,  set  forth  in  a  number  of  experimental  studies, 
would  go  even  further  than  this.  We  incline  to  the  belief  that  all 
antibodies,  including  the  so-called  amboceptors  or  sensitizers  that  take 
part  in  the  phenomena  of  lysis  and  bactericidal  action  are  essentially 
of  one  type;  that  the  fundamental  phenomenon  is  the  union  of  the  anti- 
gen with  the  specific  antibody  or  its  "sensitization;"  that  by  such  sen- 
sitization the  antigen  is  now  rendered  on  the  one  hand  more  easily 
agglutinable  or  precipitable,  on  the  other  may  be  rendered  more  amen- 
able to  the  action  of  the  alexin  or  complement  or  to  phagocytosis. 
The  agglutination  and  precipitation  phenomena,  moreover,  are  merely 
evidences  of  the  fact  that  these  substances  are  in  colloidal  suspension 
and  are  influenced  by  agencies  which  produce  precipitations  in  such 
suspension.  It  is  interesting  to  note  in  this  connection,  also,  that  bac- 
teria in  neutral  suspension  carry  negative  charges  which  can  be  weak- 
ened by  sensitization  with  serum  and  weakened  or  reversed  by  the 
addition  of  acid.    These  points  tend  to  strengthen  such  a  point  of  view. 

The  degree  of  acidity  necessary  to  reverse  the  normal  negative 
charge  of  bacteria  corresponds  roughly  to  that  at  which  growth  is  inhib- 
ited. This  has  led  me  to  speculate  whether  or  not  vitality  of  bacteria 
and  the  negative  charge  may  be  related. 

1  Neisser  and  Friedmann,  Miinch.  med.  Woch.,  1904,  li.  465-827. 


TMCnMMD 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  243 

Multiplicity  of  the  Complement. — A  number  of  very  complicated  ex- 
periments have  been  carried  out  by  Ehriich,  Morgenroth,*  Sachs,^.and 
others,  which  seem  to  show  that  the  same  serum  may  contain  a  variety 
of  complements.  Similar  conclusions  have  been  drawn  by  Wechsberg  ' 
and  by  Wassermann,*  who  demonstrated  separate  complements  for 
bactericidal  and  hemolytic  amboceptors  in  the  same  serum.  Bordet  ^ 
and  his  school,  on  the  other  hand,  deny  the  multiplicity  of  the  comple- 
ment, and,  basing  their  views  upon  numerous  experimental  data,  contend 
that  any  given  senmi  contains  but  one  alexin  or  complement.  Buchner 
and  Gruber  share  the  views  of  Bordet,  and,  in  the  light  of  recent  work, 
especially  with  complement  fixation  (see  below),  it  seems  more  likely 
that  one  and  only  one  alexin  exists  in  any  given  serum. 

Anticomplements  and  Antiamboceptors. — Antihemolytic  Action. — 
There  are  many  agencies  which  seem  to  interfere  with  the  activities  of  a 
hemolytic  system  or  of  any  antigen-antibody-alexin  complex.  The  so- 
called  anti-complements  are  many,  complement  action  being  inhibited 
by  many  non-specific  substances  such  as  bacterial  protein,  yeast,  col- 
loidal suspensions  of  acid,  colon  suspensions,  etc.  While  these  are 
chiefly  of  theoretical  interest,  there  are  other  anticomplementary  actions 
vhich  are  exerted  by  lipoids  and  globulins  of  the  senmi.  Noguchi  has 
demonstrated  a  lipoidal  substance  in  many  normal  sera  which  directly 
inhibits  the  action  of  complement  and  is  thermostable.  Browning, 
Zinsser  and  Johnson  and  others  have  shown  that  anticomplementary 
action  which  develops  in  normal  sera  on  standing  is  referable  to  the 
globuHns  of  the  sera  but  is  a  thermolabial  action  which  can  be 
removed  by  inactivation. 

Hemolytic  sera,  having  the  power  of  destroying  red  blood  cells, 
must  necessarily  prove  in  the  presence  of  sufficient  complement  to  be 
powerful  poisons  when  introduced  into  animals  whose  corpuscles  they 
are  able  to  injure.  By  careful  and  gradual  dosage  with  such  hemolytic 
sera,  Ehriich  and  Morgenroth,*  as  well  as  Bordet,^  have  been  able  to 
produce  immunity  against  the  hemolytic  action.  Thus  antihemolytic 
sera  have  been  produced,  the  action  of  which  may  depend  either  upon 
the  presence  of  anticomplement  or  of  antiamboceptor.  The  presence 
of  anticomplement  in  such  sera,  it  is  believed,  has  been  demonstrated 

'  Ehriich  unci  Margenroth,  Berl.  kiln.  Woch.,  1900. 

«  Ehriich  und  Sachs,  Berl.  klin.  Woch.,  1902.  =»  Wechsberg,  Zeit.  f .  Hyg.,  1902. 

*  Wassermann,  Zeit.  f.  Hyg.,  1901.    '  Bordet,  Ann.  de  I'inst.  Pasteur,  1900  &  1901. 

•  Ehriich  und  Margenroth,  Berl.  klin.  Woch.,  xxxi,  1900. 
">  Bordet,  Ann.  de  I'inst.  Pasteur,  t.  14,  1900. 


244  INFECTION  AND  IMMUNITY 

by  mixing  inactivated  hemolytic  serimi  with  its  respective  red  blood 
cells,  then  adding  the  antiserum  and  later  complement.  After  cen- 
trifugalization  and  separation  of  the  corpuscles,  these  may  be  dissolved 
by  the  addition  of  fresh  complement.  This  proves  conclusively  that 
there  was  no  obstacle  in  the  original  mixture  to  the  absorption  of  the 
immime  body  by  the  red  blood  cells,  and  that  the  antihemolytic  prop- 
erties of  the  serum  must  be  attributed  to  an  anticomplement.  This 
was  the  method  of  experimentation  employed  by  Ehrlich  and  Morgen- 
roth.^  Antiamboceptors  have  been  produced  by  the  same  authors  as 
well  as  by  Bordet  ^  and  Miiller,^  against  hemolytic  amboceptors. 

Complementoids. — Ehrlich  and  Morgenroth  and  Miiller  have  suc- 
ceeded in  producing  anticomplements  by  the  treatment  of  animals  with 
normal  heated  serum.  They  explain  this  by  assuming  that  the  heating 
has  not  entirely  destroyed  the  complement  in  the  normal  serimi,  but 
that  this,  analogous  to  toxin,  possesses  two  groups,  a  haptophore  and  a 
zymophore  group.  Heating  destroys  the  zymophore  without  affecting 
the  haptophore  group.  The  resulting  body,  which  corresponds  to 
toxoid,  they  call  "complementoid." 

Further  evidence  for  the  existence  of  such  complementoids  has  been 
claimed  by  Ehrlich  and  Sachs  ^  in  working  with  dog  serum.  Unheated 
dog  seriun  hemolyzes  guinea-pig  corpuscles.  Heated  to  52  degrees  C. 
for  thirty  minutes,  it  no  longer  hemolyzes  these  corpuscles  owing  to 
complement  destruction.  Such  heated  dog  serum  can  be  reactivated 
by  fresh  guinea-pig  serum  (complement).  If,  however,  the  corpuscles 
are  left  in  contact  with  the  heated  dog  blood  for  two  hours,  reactivatio^i 
by  the  guinea-pig  serum  no  longer  occurs — that  is,  the  addition  of  guinea- 
pig  seriun  no  longer  causes  hemolysis.  They  conclude  from  this  that 
the  hemolytic  amboceptor  of  the  dog  serum  has  been  attached  by  its 
complementophile  group  to  complementoids  produced  in  the  heating — 
leaving  no  point  of  attachment  for  the  complement  added  later.  These 
experiments  have  failed  of  confirmation  by  Gay  ^ — who  with  Bordet  de- 
nies the  existence  of  complementoids. 

Muir,  on  the  other  hand,  claims  to  have  demonstrated  the  existence 
of  complementoids  by  experiments  too  complicated  to  be  detailed  in 
this  place.  The  question  of  complementoids  must  be  left  undecided  until 
further  work  has  been  done. 

*  Ehrlich  und  Morgenroth,  loc.  cit. 

2  Bordet,  loc.  cit.  3  P.  Th.  Muller,  Cent.  f.  Bakt.,  1901. 

*  Ehrlich  and  Sachs,  "Ehrlich  Collected  Studies  on  Inamunity,"  trans.  byBoldnau. 

*  Gay,  Cent.  f.  Bakt.,  I,  xxxix,  1905. 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  245 

Other  Pacts  Concerning  Complement. — Muir  and  Browning  have 
shown  that,  on  the  filtration  of  serum,  amboceptor  or  immune  body 
will  pass  through  the  filter,  whereas  alexin  or  complement  is  held  back. 

This  retention  of  complement  by  filters  occurs  only  when  new  filters 
are  used,  and  it  is  our  opinion  that  this  is  probably  due  to  absorption 
or  complement  by  the  finely  divided  substances  which  make  up  the 
filter  and  not  due  to  retention  because  of  the  large  size  of  the  comple- 
ment molecule. 

Complement  can  be  inactivated  by  shaking  as  well  as  by  heat  when 
diluted  1  :  10  and  shaken  for  about  20  minutes  in  salt  solution.  Ac- 
cording to  Gramenitski  it  is  spontaneously  partially  reactivated  on 
standing. 

Complement  is  dependent  upon  the  total  volume  of  the  mixture  in 
which  it  acts,  i.e.,  upon  concentration,  the  same  actual  quantity  of 
complement  acting  more  strongly  in  higher  than  in  lower  concentrations, 
this  not  being  true  of  amboceptor  or  sensitizer  which  acts  in  direct 
proportion  to  its  actual  quantity  independent  of  the  concentration. 

Complement  is  inhibited  by  hypertonic  salt  solution  and  can  be 
preserved  in  15-25  per  cent  salt  concentration  for  weeks  in  the  icebox, 
resuming  its  activity  when  diluted  to  isotonicity  with  distilled  water. 
Removal  of  salt  by  dialysis  or  other  means  of  globulin  precipitation 
divides  the  complement  into  two  fractions,  the  globulin  fraction  and 
the  albumin  fraction,  neither  of  which  will  act  alone,  but  which  to- 
gether possess  the  properties  of  undivided  complement.  The  globulin 
fraction  attaches  directly  to  the  sensitized  cells  and  is  therefore  spoken 
of  by  German  investigators  as  "mid-piece."  The  albumin  fraction 
acts  upon  the  sensitized  gells  only  after  attachment  of  the  globulin 
fraction  and  is  therefore  spoken  of  as  *' end-piece." 

The  Fixation  of  Complement  by  Precipitates. — It  has  been  found  by 
Gengou  ^  and  confirmed  by  Moreschi,  Gay,  ^  and  others,  that  when  the 
serum  of  an  animal  immunized  with  the  serum  of  another  species  or 
with  a  foreign  albumin  is  mixed  with  a  solution  of  the  substance  used 
in  the  immunization,  the  precipitate  formed  will  remove  complement 
from  the  mixture.  In  other  words,  precipitates  formed  by  the  reaction 
of  precipitin  with  its  antigen  will  fix  complement.  This  is  of  great  im- 
portance in  complement-fixation  tests;  for  because  of  insufficient  wash- 
ing, the  blood  cells  used  in  producing  the  hemolytic  amboceptor,  may, 
from  the  presence  of  serum,  give  rise  to  a  precipitin  as  well  as  a  hemo- 

1  Gengou,  Ann.  Past.,  1902.  «  Gay,  Cent,  f .  Bakt.,  I,  xxix,  1905. 


246  INFECTION  AND  IMMUNITY 


™ 


lysin.  In  the  test  done  subsequently,  a  precipitin  reaction  may  take 
place  and  by  thus  removing  complement  may  give  a  false  result.  The 
absorption  of  complement  by  such  precipitates  takes  place  when  the  two 
reacting  factors,  the  precipitin  and  its  antigen,  are  in  dilution — so  high 
a  visible  precipitate  can  not  be  observed.  This  fact,  together  with 
others  too  complicated  to  be  discussed  in  this  place,  have  led  us  to  the 
belief  that  the  so-called  precipitins  are  true  sensitizers,  exerting  toward 
unformed  proteins  the  same  function  that  the  so-called  sensitizer  or 
amboceptor  exerts  toward  cellular  formed  antigens.     (See  p.  241.) 

Quantitative  Relationship  Between  Amboceptor  and  Complement. — 
Morgenroth  and  Sachs  ^  have  succeeded  in  showing  that  within  certain 
limits  an  inverse  relationship  exists  between  these  two  bodies.  If  for 
a  given  quantity  of  red  blood  cells  a  certain  quantity  of  amboceptor 
and  complement  suffices  to  produce  complete  hemolysis,  reduction  of 
either  the  complement  or  the  amboceptor  necessitates  an  increase  of  the 
other  factor.  As  amboceptor  is  increased,  in  other  words,  complement 
may  be  reduced  and  vice  versa.  This  result  is  of  great  importance  in 
arguing  against  the  original  conception  of  Ehrlich  in  supposing  these 
substances  to  act  together  unit  for  unit. 

Deviation  of  the  Complement  (Complement-Ablenkung). — It  was 
noticed  by  Neisser  and  Wechsberg^  that  in  mixing  together  bacteria, 
inactivated   bactericidal   immune   serum    (immune 
Hf^       T|r  yf  body),  and  complement  in  the  test  tube,  a  great 

excess  of  immune  body  hindered  rather  than  helped 
bactericidal   action.      As   the   amount  of  immune 
body    in   the    mixture    was    carried    beyond    the 
J^  H  It   experimental  optimum,  bactericidal  action  became 

less  and  less  pronounced,  and  was  finally  com- 
pletely suspended.  They  explain  this  by  assuming 
that  free  immune  body,  uncombined  with  comple- 

Dlj    n  ment,  has  a  greater  affinity  for  the  bacterial  receptor 

11    11  than   the   immune   body  combined   with    comple- 

ment.    The  complement  is  consequently  diverted 
and  prevented  from  activating  the  amboceptor  at- 
tached  to    the    bacterial    cell.      Graphically,    the 
conditions  may  be  illustrated  as  follows : 

The  above  theory  of  Neisser  and  Wechsberg  is  here  stated  simply 

^Morgenroth  und  Sachs,  "Gesammel.  Arb.  fiir  Immunitatsforschung."  Berlin, 
Hirschwald,  1904. 

2  Neisser  und  Wechsberg,  Miinch.  med.  Woch.,  xviii,  1901, 


/!/ 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  247 

because  of  the  wide  discussion  it  has  aroused.  In  the  light  of  our 
present  knowledge  concerning  the  relations  between  antigen,  ambo- 
ceptor, and  complement,  their  conception  is  obviously  erroneous. 

Fixation  of  the  Complement.— Bordet  and  Gengou^  in  1901,  devised 
an  ingenious  method  of  experimentation  by  which  even  very  small 
quantities  of  any  given  immune  body  (amboceptor)  can  be  demon- 
strated in  serum.  The  term  "fixation  of  complement,"  by  which  their 
method  of  investigation  is  now  generally  known,  explains  itself,  as  the 
steps  of  experimentation  are  followed.  They  prepared  the  following 
mixtures : 

(a)  (b) 

Bacteriolytic  amboceptor  Normal  serum,  heated 

{Plague  immune  serum,  heated) 

+  .  + 

Plague  emulsion  Plague  emulsion 

+  + 

Complement  Complement 

{Fresh  normal  serum)  {Fresh  normal  serum) 

To  both  of  these  after  five  hours  was*  added 

Hemolytic  amboceptor 

{Heated  hemolytic  serum) 

+ 

Red  blood  cells 

Results: 

(a)  showed  no  hemolysis. 

(b)  showed  hemolysis  +.  " 

The  conclusion  to  be  drawn  from  this  was  that  in  (a)  the  presence 
of  immune  body  had  led  to  absorption  of  all  the  complement.  In  (b), 
there  being  no  bacteriolytic  immune  body  to  sensitize  the  bacteria  and 
enable  them  to  absorb  complement,  the  latter  substance  was  left  free 
to  activate  the  subsequently  added  hemolytic  amboceptors.  The 
Bordet-Gengou  phenomenon  has  been  extensively  used  by  Wassermann 
and  Bruck,^  Neisser  and  Sachs,^  and  others  to  demonstrate  the  presence 
of  immune  bodies  in  various  sera.    (See  p.  262.) 

It  should  be  noted  that  this  method,  if  valid,  must  presuppose  the 
identity  of  the  hemolytic  and  bactericidal  complement  in  the  activating 
serum. 

Complement  fixation  will  be  more  extensively  discussed  in  the  sec- 
tion dealing  with  the  Wassermann  reaction. 

1  Bordet  et  Gengou,  Ann.  de  I'inst.  Pasteur,  1901. 

2  Wassermann  und  Bruck,  Med.  Klin.,  1905. 

» Neisser  und  Sachs,  Berl.  khn.  Woch.,  xliv,  1905,  and  i,  1906. 


248  INFECTION  AND  IMMUNITY 

The  Specificity  of  Hemolysins. — In  the  sections  preceding  we  have 
seen  that  the  blood  cells  of  one  animal,  injected  into  an  animal  of  an- 
other species,  give  rise  to  a  hemolytic  substance  in  the  blood  serum  of 
the  second  animal,  which  is  strictly  specific  for  the  variety  of  cells  in- 
jected. Such  hemolysins,  when  produced  in  one  animal  against  blood 
cells  of  another  species,  are  spoken  of  as  heterolysins.  In  studying  the 
nature  of  hemolysis,*  Ehrlich  and  Morgenroth  ^  now  discovered  that 
hemolysins  could  also  be  produced  if  an  animal  were  injected  with  red 
blood  cells  of  a  member  of  its  own  species.    Such  hemolytic  substances 


1^     WK.^m;^^omp\enxe-ri\ 


B 


S^pWHHc  presenf/  Togefher  at       4. 

iTnntvine  or     ?       °^       i  37.SO  C.  . 

aniibody  n.o4-      A  j»  i 

'  for  one  hour  ' 


-Haetnolyitc 
Amt  o  ce  pto  r 


(s.^g — R^d  bipod  cell 
3.   ^-•-Anfi^ert 

If   (2)    present,  no   haemolysis. 

If   (2)   not  present,   haemolysis. 

Fig.  64. — Schematic  Representation  of  Complement  Fixation  in  the 
Bordet-Gengou  Reaction. 

they  called  isolysins.  In  their  experiments  they  injected  goats  with 
the  washed  red  blood  corpuscles  of  other  goats  and  found  that  the 
serum  of  the  recipient  developed  the  power  of  causing  hemolysis  of 
the  red  blood  cells  of  the  particular  goat  whose  blood  had  been  used 
for  injection.  It  did  not,  however,  possess  the  power  of  producing 
hemolysis  in  the  blood  of  all  goats,  nor  did  it  produce  hemolysis  with  the 
red  corpuscles  of  its  own  blood.  It  is  thus  shown  that  the  specificity 
of  the  hemolysins  extends  even  within  the  limits  of  species,  and  is,  to 
a  certain  extent,  an  individual  property. 

The  production  of  autolysins,  that  is,  of  substances  in  the  blood 
serum  which  will  produce  hemolysis  of  the  individual's  own  corpuscles, 
has,  so  far,  been  unsuccessful. 

1  Ehrlich  und  Morgenroth,  Berliner  klin.  Woch.,  xxi,  1900. 


« 


LYSINS,  AGGLUTININS,  PRECIPITINS,  ETC.  249 

Ehrlich  and  Morgenroth,  in  the  course  of  these  experiments,  further- 
more succeeded  in  showing  that  the  injection  of  isolysins  into  animals 
produced  antiisolysins,  and  that  these  again  were  strictly  specific. 

The  almost  universal  failure  of  autolysin  production  has  found  no 
satisfactory  explanation.  It  is  supposed  by  Ehrlich  and  Morgenroth 
that  the  failure  of  autolysin  production  may  be  due  to  a  lack  of 
suitable  receptors  in  the  animal  for  its  own  cells. 

The  cHnical  significance  of  the  presence  of  isolysins  and  possibly  of 
autolysins  in  human  beings,  is  too  evident  to  require  much  discussion. 
A  practical  and  extremely  interesting  result  which  these  investigations 
have  yielded  is  that  of  Donath  and  Landsteiner,^  who  discovered  an 
autolysin  in  the  blood  serum  of  patients  suffering  from  paroxysmal 
hemoglobinuria.  In  these  cases  the  sensitizing  substance  or  ambo- 
ceptor appeared  to  be  absorbed  by  the  red  blood  cells  only  at  low  tem- 
peratures— probably  in  the  capillaries  during  exposure  to  the  cold,  and 
hemolysis  subsequently  resulted  in  the  blood  stream  by  the  action  of 
complement.  These  observations  have  been  confirmed  by  other  writ- 
ers, but  the  phenomenon  is  surely  not  present  in  all  cases  of  paroxysmal 
hemoglobinuria.  The  writers  have  had  occasion  to  examine  carefully 
several  clinically  typical  cases  with  negative  results. 

*  Donath  und  LavdsteiTwr,  Miinch.  med.  Woch.,  xxxvi,  1904. 


n 


CHAPTER   XVI 
THE  TECHNIQUE  OF  SERUM  REACTIONS 

Obtaining  Serum  from  Animals  and  Man. — To  obtain  blood  serum 
from  man,  the  blood  may  be  taken  from  the  finger  or  the  ear,  either 
into  a  sterile  centrifuge  tube  or  into  a  Wright  capsule.  When  taken 
into  a  centrifuge  tube,  the  blood  is  allowed  to  clot  and  the  serum  sep- 
arated by  centrifugation.  Larger  quantities  of  blood  may  be  taken 
with  a  syringe  from  the  median  basilic  vein  and  either  slanted  in  sterile 
test  tubes  in  the  ice  chest  or  put  into  centrifuge  tubes  and  centrifugalized. 
In  bleeding  small  laboratory  animals,  a  number  of  methods  may  be 
employed,  depending  upon  the  quantity  of  serum  required. 

The  animals  most  frequently  used  for  laboratory  purposes  are  rab- 
bits. To  obtain  small  quantities  of  serum  from  rabbits,  the  animals 
may  be  bled  from  the  marginal  vein  of  the  ear.  The  animal  is  strapped 
upon  a  tray  and  underneath  it  is  placed  a  rubber  bag  filled  with  warm 
water.  This  is  advised  by  Wadsworth  to  facilitate  the  flow  of  blood. 
The  tray  is  then  placed  upon  an  easel  so  that  the  animal's  head  hangs 
downward.  The  skin  over  the  ear  vein  is  shaved  and  sterilized,  and  a 
Hagedorn  needle  plunged  into  the  vein.  The  blood  is  caught  in  test 
tubes  or  centrifuge  tubes. 

When  larger  quantities  of  blood  are  desired  it  may  be  taken  from 
the  carotid  artery.  In  rabbits,  the  carotid  may  be  found  lying  just 
lateral  to  the  trachea  and  deeply  placed,  and  must  be  carefully  separated 
from  the  pneumogastric  nerve  by  blunt  dissection.  The  distal  end  of 
the  artery  is  then  tied  off  and  the  proximal  end  temporarily  closed 
with  a  small  clamp.  The  artery  is  then  raised  out  of  the  wound  on  a 
knife  or  forceps  handle  and,  with  sharp-pointed  scissors,  a  small  in- 
cision is  made  into  but  not  through  the  vessel.  A  small  glass  cannula 
is  now  introduced  and  tied  into  place  by  a  thread.  To  this  cannula  a 
small  rubber  tube  fitted  with  a  pinch-cock  should  have  been  attached, 
the  whole  being  sterilized.  Recently  we  have  dispensed  with  the  can- 
nula, simply  holding  the  vessel  up  with  a  pointed  forceps.  A  larger 
yield  of  serum  will  be  obtained  if,  after  coagulation,  the  clot  is  sep- 
arated from  the  glass  with  a  sterile  platinum  wire. 

250 


THE  TECHNIQUE  OF  SERUM  REACTIONS  251 

In  obtaining  blood  from  larger  animals,  horses,  sheep,  etc.,  a  cannula 
rmay  be  introduced  into  the  jugular  or  internal  saphenous  veins.  The 
skin  is  shaved  and  sterilized  and  a  rubber  tourniquet  placed  about  the 
neck  or  thigh,  as  the  case  may  be,  in  order  to  cause  the  vein  to  stand 
out.  A  small  incision  may  be  made  through  the  skin  over  the  vein,  but 
is  not  necessary.  A  cannula,  with  rubber  tubing  attached,  is  then 
plunged  into  the  vein  and  the  blood  caught  in  sterile  high  cylindrical 
jars,  allowed  to  clot,  and  placed  in  the  refrigerator.  The  serum  is  taken 
off  after  twenty-four  to  forty-eight  hours  with  sterile  pipettes. 

Agglutination  Tests.— For  the  determination  of  the  agglutinating 
power  of  serum  it  is  necessary  to  make  suitable  dilutions  of  the  serum, 
and  to  prepare  an  even  emulsion  of  the  microorganisms  to  be  tested. 
The  test  may  be  made  microscopically  or  macroscopically.  The  micro- 
scopic test  is  the  one  in  general  use  in  the  diagnosis  of  typhoid  fever, 
and  is  occasionally  applied  to  some  other  diseases.  In  its  application 
to  typhoid  fever  it  is  usually  spoken  of  as  the  Gruber-Widal  reaction. 

Twelve-  to  eighteen-hour  broth  cultures  of  the  typhoid  bacillus, 
grown  at  incubator  temperature,  may  be  used.  It  is  preferable,  how- 
ever, to  use  an  emulsion  of  a  twelve  to  twenty-four  hour  old  agar  culture 
in  physiological  salt  solution  (0.85  per  cent).  The  salt-solutron  emulsion 
is  made  by  adding  about  10  c.c.  of  normal  salt  solution  to  the  fresh  agar 
slant  culture,  carefully  detaching  the  culture  from  the  surface  of  the 
agar  with  a  flexible  platimma  wire,  and  pipetting  off  the  emulsion  thus 
made.  With  some  microorganisms  it  is  sufficient  simply  to  allow  the 
larger  clumps  to  settle  and  to  pipette  off  the  supernatant  turbid  emulsion. 
With  other  microorganisms,  the  tendency  to  form  clumps  makes  it 
necessary  to  resort  to  further  methods  of  securing  an  even  distribution 
of  the  bacteria.  This  may  be  done  either  by  sucking  the  emulsion  in  and 
out  through  a  narrow  pipette  held  perpendicularly  against  the  bottom  of 
a  watch  glass,  as  in  Wright's  technique  for  the  opsonic  test  (see  section 
on  Opsonins,  p.  285),  or  by  carefully  rubbing  the  climips  against  the 
watch  glass  with  a  stiff  platimma  wire.  In  the  case  of  the  tubercle  ba- 
cillus not  even  this  suffices,  but  it  becomes  necessary  to  grind  the  moist 
bacillary  masses  in  a  mortar  before  emulsifying.  With  the  tubercle  bacil- 
lus, too,  it  is  preferable  to  use  salt  solution  at  1.5  per  cent  concentration. 
In  preparing  cultures  of  streptococcus  and  pneumococcus  for  ag- 
glutination tests,  it  has  been  found  convenient  by  Hiss  to  grow  for 
about  four  days  in  flasks  of  a  1%  glucose,  2%  pepton  meat-infusion 
broth,  to  which  has  been  added  1%  of  calcium  carbonate  (p.  126). 
The  calcium  neutralizes    the  inhibiting    acid  formed  in    the    broth 


252  INFECTION  AND  IMMUNITY 

by  the  microorganisms  and  permits  the  development  of  a  mass  culture. 
The  flasks  should  be  shaken  at  least  once  a  day.  The  broth  may  be 
pipetted  off  and  clumps  removed  by  centrifugation.  Without  this  tech- 
nique it  is  sometimes  difficult  to  get  sufficient  growths  of  these  bacteria 
for  any  quantity  of  emulsion  unless  large  surfaces  of  agar  are  employed 
in  special  receptacles  or  by  making  many  slant  cultmes. 

The  serum  dilutions  are  obtained  by  first  making  a  one  to  ten  dilu- 
tion of  serum  with  normal  salt  solution.  The  serum  used  for  this  pur- 
pose is  of  red  blood  corpuscles  by  centrifugation.  From  the  1  to  10 
dilution  any  number  of  higher  dilutions  may  be  made,  by  mixing  given 
parts  of  the  1  to  10  dilution  with  normal  salt  solution;  thus  one  part 
of  a  1  to  10  dilution  plus  an  equal  quantity  of  salt  solution  gives  a  dilu- 
tion of  1  to  20.  One  part  of  one  to  ten  dilution  plus  two  parts  of  normal 
salt  solution  gives  one  to  thirty,  etc.  It  must  not  be  forgotten  that, 
when  equal  parts  of  the  serum  and  bacillary  emulsion  have  been  mixed, 
each  one  of  these  dilutions  is  doubled. 

In  making  the  microscopic  agglutination  test,  equal  quantities  of 
serum  dilution  and  bacterial  emulsion  are  mixed  upon  a  cover-slip. 
The  mixture  may  be  made  either  by  measuring  out  a  drop  of  each  sub- 
stance with  a  standard  platimun  loop,  depositing  them  close  together 
on  the  cover-slip,  and  mixing;  or  equal  quantities  may  be  sucked  up, 
each  to  a  given  mark,  in  a  capillary  pipette,  mixed  by  suction  in  and 
out,  and  deposited  upon  the  cover-slip.  The  cover-slip  is  inverted  over 
a  hollow  glass  slide,  the  rim  of  which  has  been  greased  with  vaseline. 
The  drop  is  then  observed  through  a  (Leitz)  No.  7  lens,  ocular  No.  3. 
Macroscopic  agglutination,  preferable  for  exact  laboratory  research, 
is  made  in  narrow  test  tubes  measuring  about  0.5  cm.  in  diameter  and 
about  5  cm.  in  length  (Fig.  60). 

Equal  quantities,  usually  1  c.c.  each,  of  serum  dilution  and  emulsion 
are  mixed.  A  series  of  tubes  is  prepared,  in  each  subsequent  one  of 
which  the  dilution  is  higher.  These  mixtures  may  be  placed  in  the  in- 
cubator for  a  few  hours  and  then  kept  at  room  temperature.  After  re- 
moval from  the  incubator  agglutination  is  in  some  instances  hastened 
by  transference  to  the  ice  chest.  When  agglutination  takes  place  in 
these  tubes,  clumps  of  bacteria  may  be  seen  to  form,  which  settle  to 
the  bottom  of  the  tube,  very  much  like  snow-flakes.  The  surface  of 
the  sediment  is  heaped  up  and  irregular.  The  supernatant  fluid  becomes 
entirely  clear.  When  the  reaction  does  not  occur  the  sediment  is  an  even, 
granular  one  with  a  flat  surface,  and  the  emulsion  remains  turbid. 

Instead  of  using  test  tubes  as  described  above,  Wright  has  sug- 


THE  TECHNIQUE  OF  SERUM  REACTlONg  255 

gested  the  us6  of  throttle  pipettes  of  comparatively  large  diameter  into 
each  of  which  at  least  three  or  four  different  dilutions  can  be  sucked 
with  a  nipple,  a  small  air  bubble  being  left  between  the  mixtures.  By- 
sealing  the  distal  end  of  these  pipettes  in  a  flame  the  various  dilutions 
are  kept  at  a  distance  from  each  other,  and  the  pipettes  may  be  set  on 
end  in  a  tumbler  and  observed  just  as  are  the  test  tubes  (Fig.  68,  p.  285). 
The  special  methods  of  carrying  out  agglutination  tests  with  pneumo- 
cocci  have  been  described  on  p.  363. 

Precipitin  Tests. — In  an  earlier  section  on  precipitins  we  have  seen 
that  precipitates  are  formed  when  clear  filtrates  of  bacterial  extracts 
or  of  both  cultures  are  mixed  with  their  specific  immune  sera.  Such 
precipitin  reactions  are  not  limited  to  the  realm  of  bacteria,  but  have  a 
broad  biological  significance,  in  that  specific  precipitating  sera  may  be 
produced  with  proteids  of  varied  source. 

For  carrying  out  a  precipitin  test,  the  following  reagents  are  required: 

1.  A  specific  precipitating  antiserimi  (antibacterial  or  antiproteid) ; 

2.  A  bacterial  filtrate  or  proteid  solution. 

Production  of  Precipitating  Antisera.' — Antibacterial  precip- 
itins may  be  produced  in  animals  by  a  variety  of  methods.  Animals, 
preferably  rabbits,  are  injected  with  cultures  of  the  bacteria  in 
gradually  increasing  quantities.  Five  or  six  injections  are  given  at 
intervals  of  from  five  to  six  days,  the  dosage  and  mode  of  administra- 
tion being  adapted  in  each  case  to  the  pathogenicity  of  the  micro- 
organisms in  question.  Myers  ^  claims  that  specific  precipitin  for  pepton 
in  the  culture  media  may  be  formed  which  may  lead  to  error.  This 
could  not  be  confirmed  by  Norris.^  The  immunized  animals  should 
be  bled  about  7  to  12  days  after  the  last  injection. 

Precipitating  antisera  against  protein  solutions  are  prepared  by 
similar  methods.  The  sera  or  protein  solutions  used  should  be  sterile. 
This  may  be  accomplished  by  filtration  through  small  porcelain  filters. 
Injections  into  animals  may  be  made  subcutaneously,  intraperitoneally, 
or  intravenously.  The  subcutaneous  route  has  no  advantages  unless 
the  substances  to  be  used  are  contaminated. 

Nuttall  advises  the  use  of  rabbits.  The  animals  are  weighed  from 
time  to  time,  and  if  considerable  loss  of  weight  ensues,  the  intervals 
should  be  increased.  Doses  from  2  to  5  c.c.  should  be  given.  In 
giving  the  later  injections  the  danger  of  anaphylaxis  must  be  remem- 


1  R.  Kraus,  Wien.  klin.  Woch.,  1897;  Norris,  Jour.  Inf.  Dis.,  1  and  3,  1904. 
^  Myers,  Lancet,  ii,  1900.     ^  Norris,  loc.  cit. 


^54  INFECTION  AND  IMMUNITY 

bered.  A  single  injection  of  a  large  quantity  has  occasionally  yielded 
a  precipitating  serum  of  considerable  strength,^  but  this  method  is 
not  usually  successful.  Injections  are  made  at  intervals  of  from  five  to 
seven  days.  Seven  to  twelve  days  after  the  last  injection  the  animals 
may  be  bled  from  the  ear,  and  a  preliminary  test  made  to  ascertain  the 
precipitating  value  of  the  serum.  If  this  is  insufficient,  more  injections 
may  be  made.  Bleeding  should  be  done  7  to  12  days  after  the  last 
injection.  Such  sera  may  be  preserved  in  the  dark  and  at  a  low  tem- 
perature. If  a  preservative  is  added,  Nuttall  prefers  chloroform  to 
the  phenols,  because  of  occasional  turbidity  produced  by  these. 

Precipitating  antisera  for  tests  should  be  clear.  If  turbid,  the  sera 
should  be  filtered  through  small  Berkefeld  or  porcelain  candles. 

Preparation  of  Bacterial  Filtrates  and  Protein  Solutions 
FOR  Precipitin  Tests. — Bacteria  may  be  grown  in  nutrient  broth 
having  an  initial  reaction  of  neutrality  or  five-tenths  per  cent  acidity 
to  phenol ophthalein.  The  cultures  are  incubated  for  times  varying  from 
a  week  to  several  months,  and  are  then  filtered  through  porcelain  or 
Berkefeld  candles  until  perfectly  clear.  Bacterial  extracts  may  also 
be  made  by  emulsifying  agar  cultures  in  salt  solution,  placing  at  37.5°  C. 
in  the  incubator  for  a  week  or  longer,  and  filtering.  More  rapid  ex- 
traction of  bacteria  may  be  accomplished  by  repeated,  rapid  freezing 
and  thawing  of  salt-solution  emulsions,  by  shaking  in  the  shaking  ma- 
chine or  by  centrifugalizing,  rubbing  up  the  sediment  with  dry  salt, 
and  the  addition  of  distilled  water  to  isotonicity. 

Protein  solutions  to  be  tested  should  be  made  in  salt  solution.  When 
dealing  with  blood  stains,  as  in  doing  the  test  for  forensic  purposes, 
the  stains  should  be  dissolved  in  salt  solution,  an  approximate  dilution 
of  one  in  five  hundred  being  aimed  at.  This  solution  if  turbid  should 
be  filtered  through  a  small  porcelain  filter.  It  should  be  clear  and  color- 
less, show  a  faint  cloud  on  boiling  with  dilute  acetic  acid,  and  show 
distinct  froth  when  shaken. 

When  the  reaction  is  to  be  done  for  determining  the  nature  of  meat 
(detection  of  horse-meat  substitution  for  beef,  etc.),  about  20  to  40 
grams  of  the  suspected  meat  are  macerated  in  a  flask,  and  covered  with 
100  c.c.  of  salt  solution.  This  mixture  is  allowed  to  infuse  at  room 
temperature  for  three  to  four  hours,  and  is  then  placed  in  the  refrigerator 
for  twelve  hours  or  more.  At  the  end  of  this  time  2  c.c.  are  shaken 
into  a  test  tube.    If  profuse  frothing  ^  appears,  the  extract  is  ready  for 

1  Michaelis,  Deut.  med.  Woch.,  1902. 

2  P.  Th.  Muller,  "Technik.  d.  serodiagnos.  Methoden." 


THE  TECHNIQyE  OF  SERUM  REACTIONS  255 

use.  It  is  then  filtered  clear,  either  through  paper,  or  through  a  Buchner 
or  Nutsche  filter.  Berkefeld  filters  may  also  be  used.  The  solution  is 
then  diluted  until  the  addition  of  concentrated  HNO3  produces  only  a 
slight  even  turbidity.  Before  use  the  reaction  of  the  meat  extract 
should  be  tested,  and  if  necessary  adjusted  to  neutrality  or  slight  acidity 
or  alkalinity. 

In  the  actual  test  with  bacterial  filtrate,  the  procedure  is  as  follows: 
In  a  series  of  narrow  test  tubes,  the  following  mixtures  are  made: 

Tube  1.  Antibacterial  serum  .5  c.c.  +  bacterial  filtrate  1.  c.c. 
"      2.  Normal  serum  .5  c.c.  +  bacterial  filtrate  1.  c.c. 

"     3.  Antibacterial  serum  .5  c.c.  +  salt  solution         1.  c.c. 
"     4.  Salt  solution  .5  c.c.  +  bacterial  filtrate  1.  c.c. 

Place  the  tubes  in  the  incubator  at  37.5°  C.  Tube  1  only  should 
.show  a  haziness  which  develops  into  distinct  cloudiness  or  a  flocculent 
precipitate  within  one  hour.    Tubes  2,  3,  and  4  should  remain  clear. 

In  testing  an  unknown  protein  with  serum  of  an  animal  immunized 
with  the  protein  sought  for,  the  technique  of  the  test  is  as  follows: 

1.  0.1  c.c.  immune  serum  +  2  c.c.  unknown  protein  solution. 

2.  0.1  c.c.  immune  serum  +  2  c.c.  known  protein  solution  of  variety  suspected 

(similarly  diluted). 

3.  0.1  c.c.  immune  serum  +  2  c.c.  protein  solution  of  different  nature  (similarly 

diluted) . 

4.  0.1  c.c.  immune  serum  +  2  c.c.  salt  solution. 

5.  2  c.c.  unknown  protein  solution. 

If  the  test  is  positive  a  precipitate  appears  in  tubes  1  and  2,  but  not 
in  any  of  the  others.  The  precipitate  should  appear  within  15  to  20 
minutes. 

Bactericidal  and  Bacteriolytic  Tests. — The  bactericidal  and  bac- 
teriolytic powers  of  serum  may  be  tested  either  in  the  animal  body  or 
in  the  test  tube.  The  in  vivo  test  is  known  as  Pfeiffer's  phenomenon. 
This  depends  upon  the  fact  that  bacteria,  when  injected  into  the  peri- 
toneal cavity  of  a  guinea-pig,  together  with  a  homologous  immune 
serum,  undergo  dissolution. 

As  practiced,  the  test  finds  a  double  application.  It  may  be  done  to 
determine  the  bacteriolytic  power  of  a  given  serum  against  a  known 
microorganism,  or  for  the  identification  of  a  particular  microorganism 
by  means  of  its  susceptibility  to  lysis  in  a  known  immune  serum. 

1.  Determination  of  the  bacteriolytic  power  of  serum  against  a  known 
microorganism  in  vivo:  ^ 

^  P.  Th.  Muller,  "Technik  d.  serodiagnos.  Methoden,"  Jena,  1909. 


256  INFECTION  AND  IMMUNITY 

A  number  of  dilutions  of  the  serum  are  made  with  sterile  neutral 
bouillon  or  salt  solution,  ranging  from  1  in  20  to  1  in  500,  or  higher.  It 
is  convenient  to  make  a  first  solution  of  1  in  20.  One  c.c.  of  this  mixed 
with  4  C.C.  of  broth  will  give  1  in  100.  One  c.c.  of  the  1  in  100  dilution 
with  1  c.c.  of  broth,  2  c.c.  of  broth  and  4  c.c.  of  broth  will  give  1  in  200, 
1  in  300,  and  1  in  500,  respectively.  Into  one  cubic  centimeter  of  each 
of  these  dilutions  there  is  placed  one  platinum  loopful  of  a  twenty-four- 
hour  agar  culture  of  the  microorganism  against  which  the  serum  is  to 
be  tested.  Into  another  test  tube  is  placed  4  c.c.  of  broth,  without 
serum,  and  with  one  loopful  of  the  microorganisms.  The  mixtures 
are  thoroughly  emulsified  in  each  case  by  rubbing  the  bacteria  against 
the  sides  of  the  tube  with  the  platinum  loop. 

Intraperitoneal  injections  into  guinea-pigs  are  then  made  of  1  c.c. 
of  each  of  the  serum-dilution-bacterial-emulsions.  A  control  guinea-pig. 
(better  two  or  three)  receives  Ic.  c.  of  the  broth  emulsion — one-fourth 
as  many  bacteria,  therefore,  as  the  animals  receiving  the  serum 
dilutions. 

Before  making  the  injections,  areas  on  the  lateral  abdominal  walls 
of  the  guinea-pigs  are  shaved,  and  small  incisions  made  through  the 


1 


Fig.  65. — Capillary  Pipette  for  Removal  of  Exudate  in  doing  the 

Pfeiffer  Test. 

skin,  down  to  the  muscular  layers.  The  needle  of  the  syringe  is  then 
introduced  perpendicular  to  the  skin  until  it  has  penetrated  the  peri- 
toneum, and  then  carefully  slanted  to  avoid  puncturing  the  gut.  The 
animals  need  not  be  strapped  down  during  this  procedure  and  after- 
ward may  be  allowed  to  run  about. 

After  one-half  hour,  and  again  after  one  hour  has  elapsed,  a  drop 
of  peritoneal  exudate  is  removed  from  each  guinea-pig  and  examined 
in  the  hanging  drop  for  granulation  and  swelling  of  the  bacteria.  The 
method  of  obtaining  the  peritoneal  exudate  is  as  follows:  Small  glass 
tubing  is  drawn  out  into  capillary  pipettes,  the  ends  of  the  capillaries 
being  again  drawn  to  fine  points  in  a  small  yellow  flame.  A  number  of 
such  pipettes  should  be  prepared  before  the  test  is  begun.  The  guinea- 
pig  is  then  held  down  upon  a  table,  either  by  an  assistant  or  by  the  left 
hand  of  the  operator,  and  the  point  of  the  pipette  pushed  through  the 
ent  in  the  abdominal  wall  into  the  peritoneum  by  a.  sharp,  quick  thrust- 


THE  TECHNIQUE  OF  SERUM  REACTIONS  257 

ing  motion.  A  column  of  peritoneal  fluid  will  run  into  the  glass  tubing 
by  capillary  attraction;  this  can  then  be  blown  out  upon  a  cover-slip 
for  hanging-drop  examination  or  may  be  blown  upon  a  sHde,  smeared, 
and  examined  after  staining.  The  reaction  is  regarded  as  positive  if 
within  thirty  minutes  to  an  hour  the  peritoneal  exudates  of  the  animals 
receiving  immune  sera  contain  only  swollen  or  disintegrated  microor- 
ganisms, while  in  that  of  the  control  animals  only  well-preserved  and 
undegenerated  bacteria  are  found.  In  dealing  with  typhoid  bacilli 
and  cholera  spirilla,  in  connection  with  which  the  test  is  most  often 
used,  active  motility  in  the  controls  is  of  much  help.  Should  there  be 
extensive  degeneration  of  the  bacteria  in  the  exudate  of  the  control 
animals  the  test  is  of  no  value. 

2.  Identification  of  a  microorganism  by  observing  its  a  susceptibility  to 
lysis  in  a  known  immune  serum  in  vivo: 

The  technique  for  this  test  is  practically  the  same  as  that  of  the 
preceding  except  that  in  this  case  we  require  a  potent  known  immune 
serum  and  normal  serum  for  control.  It  is  necessary,  furthermore,  that 
by  previous  tests  we  should  know  the  degree  of  dilution  in  which  the 
immune  serum  will  cause  complete  bacteriolysis  of  the  microorganism 
used  in  its  production.  Thus,  if  we  are  employing  a  typhoid  immune 
serum  and  are  about  to  test  by  this  method  an  unknown  Gram-negative 
bacillus,  we  must  know  the  titer  of  the  serum  for  the  typhoid  bacillus 
itself. 

Mixtures  are  then  made  of  dilutions  of  this  serum  and  definite 
quantities  of  the  microorganism  to  be  tested.  It  is  best,  always,  to 
employ  from  ten  to  one  hundred  times  the  amount  of  immune  serum 
which  suffices  to  produce  lysis  with  its  homologous  microorganism. 
Thus,  if  the  serum  has  been  found  to  be  active  in  dilutions  of  1  : 1 ,000,  it 
is  employed  in  the  test  in  dilutions  of  1  : 1,000, 1  :  100,  and  1  :  10.  These 
dilutions  are  then  injected  into  guinea-pigs  in  quantities  of  1  c.c.  together 
with  the  bacteria  to  be  tested,  and  control  guinea-pigs  are  injected  with 
undiluted  normal  serum  mixed  with  the  bacteria  and  with  salt  solution 
and  the  bacteria.  The  exudates  are  then  observed  in  the  same  way  as 
in  the  preceding  experiment. 

Bactericidal  Reactions  in  the  Test  Tube. — Bactericidal  reactions 
in  the  test  tubes  may  be  made  by  mixing  in  small  sterile  test  tubes, 
definite  quantities  of  the  bacteria  with  inactivated  s^rum  and  com- 
plement, the  latter  in  the  form  of  unheated  normal  serum.  The 
mixtures,  diluted  with  equal  volumes  of  neutral  broth  or  salt  solution, 
are  set  away  for  a  definite  time  three  to  four  hours  in  an  incubator  at 


258  INFECTION  AND   IMMUNITY 

37.5°  C,  and  equal  quantities  from  all  the  tubes  are  then  inoculated 
into  melted  agar  at  40°  C,  and  plates  are  poured.  Control  plates 
must  be  made  in  each  case  with  mixtures  of  similar  quantities  of 
bacteria  in  salt  solution,  and  similar  quantities  of  bacteria  in  normal 
serum.  By  colony  counting  after  the  plates  have  developed,  it  is  then 
possible  to  estimate  the  degree  of  bacterial  destruction  in  any  of  the 
given  dilutions. 

In  actually  carrying  out  the  test,  dilutions  of  the  inactivated  serum 
are  first  made,  ranging  from  1  :  10  to  1  :  1,000  and  over.  An  emulsion 
of  bacteria  from  a  twenty-four-hour  agar  slant  is  then  made  in  salt  solu- 
tion, or  a  twenty-four-hour  broth  culture  properly  diluted  may  be  used. 
Complement  is  obtained  by  taking  fresh  normal  rabbit  serum  and  dilut- 
ing it  with  salt  solution  1  :  10  or  1  :  15.  Into  a  series  of  test  tubes,  then, 
1  c.c.  of  each  of  the  serum  dilutions  is  placed,  and  to  each  tube  is  added 
0.5  c.c.  of  the  diluted  fresh  normal  rabbit  serum  (complement) .  To  these 
mixtures  the  bacteria  are  then  added.  In  adding  the  bacterial  emulsion 
to  these  tubes,  the  writers  have  found  it  more  accurate  to  discard  the 
use  of  the  platinum  loop  and  to  measure  the  bacterial  emulsion  in  a 
marked  capillary  pipette  such  as  that  used  in  the  opsonin  test.  (See  page 
285,  Fig.  68.)  The  controls  are  set  up  in  a  similar  way,  all  of  them  con- 
taining a  similar  quantity  of  bacterial  emulsion,  one  control  containing 
1.5  c.c.  of  salt  solution,  another  control  containing  1  c.c.  of  salt  solution  + 
0.5  c.c.  of  the  diluted  complement,  and  the  third  control  containing  in- 
activated normal  serum  1  c.c. +0,5  c.c.  of  diluted  complement.  Defi- 
nite quantities  of  these  mixtures,  taken  with  a  standard  loop,  or 
preferably  with  a  capiUary  pipette,  are  plated  in  agar  immediately 
after  mixing. 

BACTERICIDAL   TEST    IN    VITRO 

(To  Determine  the  Bactericidaij  Power  of  a  Typhoid  Immune  Serum 
AGAINST  Typhoid  Bacilli). 

Plates 

Poured 

After  3  Hrs. 

at  37°  C. 


.c. Immune Typh. Ser.  1 : 200    +0.5  c.c.  Typh.  Emulsion +0.5 c.c.  Rab.Ser.  1:15  ?         0 
"^       ••    1:400     +0.5    "       '^  "        +0.5   "       "      "      "     f    Colo 


1:800     +0.5  "  "  "  +0.5   " 

1:1600   +0.5  '•  "  '•  +0.5    ' 

1:3200  +0.5  "  "  "  .     +0.5    " 

1:6400  +0.5  "  "  "  +0.5    " 

1:12800  +  0.5  "  "  "  +0.5   " 

1:25600  +  0.5  "  "  "  +0.5   *• 

Controls 


Colonies. 

100-1,000 
Colonies 

More  than 

10,000 

Colonies 


la  ( 1.5  c.c.  NaCl+0.5  Typh.  Emulsion  Plated  immediately)  More  than 

-^1.5    "       "    +0.5      "  "  "       after  3  hrs.    >      10,000 

IIb(l.      "       "    +0.5     "  "        +0.5c.c.Rab.  Ser.  1:15     "  "     "    "     )    Colonies 


THE  TECHNIQUE  OF  SERUM   REACTIONS  259 

After  Incubation  for  two  or  three  hours  similar  quantities  are  again 
measured  into  tubes  of  melted  agar  with  the  capillary  pipette.  With  a 
little  practice,  great  accuracy  in  these  measurements  can  be  acquired. 
The  inoculated  agar  tubes  are  very  thoroughly  mixed,  and  plates  are 
poured.  At  the  end  of  twenty-four  hours'  incubation,  an  enumeration 
of  the  colonies  in  the  various  plates  is  made  and  the  results  are  compared. 

The  in  vitro  bactericidal  tests  have  been  employed,  practically,  chiefly 
in  the  diagnosis  of  typhoid  fever  by  Stern  and  Korte.^  While  the  serum 
of  normal  individuals  shows  practically  no  bactericidal  power  for 
typhoid  bacilli,  the  sera  of  typhoid  patients  may  be  actively  bacteri- 
cidal in  dilutions  as  high  as  1  :  50,000. 

Hemolytic  Tests. — Determination  of  the  hemolytic  action  of  blood 
serum,  bacterial  filtrates,  and  of  a  variety  of  other  substances,  such  as 
tissue  extracts  and  animal  and  plant  poisons,  is  frequently  made  in 
bacteriological  laboratories.  FamiUarity  with  the  methods  of  carrying 
out  such  tests  is  especially  essential  since  hemolytic  tests  are  also  em- 
ployed in  determining  other  serum  reactions,  such  as  the  "  complement- 
fixation  tests  "  discussed  in  another  section. 

For  these  tests  it  is  necessary  to  prepare  washed  red  corpuscles 
of  the  species  of  animal  against  which  the  hemolysins  are  to  be 
tested,  and  to  obtain  these,  blood  may  be  taken  in  one  of  the 
following  ways: 

A.  If  small  quantities  of  blood  corpuscles  are  desired,  the  blood  may 
be  received  into  a  sterile  test  tube  into  which  a  copper  or  other  wire  bent 
into  a  loop  at  the  lower  end  has  been  introduced.  This  is  used  to 
prevent  clotting  and  to  remove  the  fibrin.  Immediately  after  receiving 
the  blood  into  this  tube,  the  wire  is  twirled  between  the  fingers  so  that 
the  blood  is  beaten  by  the  wire  as  by  an  egg-beater.  At  the  end  of  five 
minutes  of  continuous  agitation,  the  fibrin  adhering  in  a  mass  to  the  wire 
may  be  lifted  out.  The  corpuscles  are  then  washed  and  centrifugalized 
in  several  changes  of  salt  solution  to  remove  all  traces  of  serum,  and 
are  finally  emulsified  in  salt  solution. 

B.  The  blood  may  be  taken  into  a  centrifuge  tube  and  immediately 
centrifugalized  before  clotting  has  taken  place.  The  plasma  is  then 
poured  off  and  the  corpuscles  are  washed  with  salt  solution,  as  before, 
to  remove  the  serum. 

C.  The  blood  may  be  taken  directly  into  a  solution  containing 
five-tenths  per  cent  sodium  chlorid  and  one  per  cent  sodium  citrate. 

»  Stem  und  Korte,  Berl.  klin.  Woch.,  1904. 


260  INFECTION  AND  IMMUNITY 

The  corpuscles  are  concentrated  by  centrifugalization,  the  citrate  solu- 
tion is  decanted,  and  corpuscles  are  washed  with  salt  solution,  as  before, 
to  remove  the  serum. 

D.  When  large  quantities  of  blood  are  desired,  either  from  man  or 
from  an  animal,  the  blood  may  be  received  directly  into  a  flask  into 
which  a  dozen  or  more  glass  beads  or  short  pieces  of  glass  tubing  have 
been  placed.  The  flask  is  shaken  for  five  or  ten  minutes,  immediately 
after  the  blood  has  been  taken  and,  in  this  way,  defibrination  is  accom- 
plished. 

Since,  for  comparative  tests,  it  is  necessary  to  establish  some  stand- 
ard concentration  of  red  blood  cells,  it  is  customary  in  these  tests  to 
employ  a  five  per  cent  emulsion  of  corpuscles  in  salt  solution.  To 
obtain  this,  one  volume  of  sediment  of  washed  red  blood  cells  is  mixed 
with  nineteen  parts  of  0.85  per  cent  salt  solution.^  Such  an  emulsion,  if 
kept  sterile  and  in  the  refrigerator,  will  serve  for  hemolytic  tests  for 
from  one  to  three  days.  An  emulsion  should  not  be  used  if  the  super- 
natant salt  solution  shows  any  transparent  redness,  as  this  indicates 
hemolysis. 

If  the  substance  in  which  hemolysins  are  to  be  determined  is  serum, 
this  should  be  inactivated  by  exposure  to  56°  C.  in  a  water  bath,  and 
to  each  test,  complement  may  be  added  in  the  form  of  fresh  guinea-pig 
or  rabbit's  serum.  No  absolute  rule  for  the  quantity  of  complement 
to  be  used  in  these  tests  can  be  given.  In  each  case  the  particular 
complement  used  should  be  titrated  to  determine  the  minimum  quan- 
tity which  will  produce  hemolysis  of  1  c.c.  of  the  sensitized  cell  sus- 
pension. 

In  the  actual  test,  mixtures  are  made  of  the  corpuscle  emulsion,  the 
inactivated  immune  serum,  and  complement  in  small  test  tubes  and  the 
volumes  of  the  various  tubes  made  equal  by  the  addition  of  definite 
quantities  of  salt  solution.  The  contents  of  the  tubes  are  thoroughly 
mixed  and  the  tubes  put  in  the  incubator  or  in  a  water  bath  at  37.5°  C. 
If  complete  hemolysis  occurs,  the  fluid  in  the  tube  will  assume  a  deep 
Burgundy  red.  If  no  hemolysis  occurs,  the  fluid  will  remain  uncolored 
and  the  corpuscles  wiU  settle  out.  Incomplete  hemolysis  will  be  evi- 
denced by  a  lighter  tinge  of  red  in  the  tube  and  by  the  settling  out  of  a 
varying  quantity  of  blood  corpuscles. 


*  The  method  here  given  was  formerly  much  employed.  It  is  now  the  general 
practice,  however,  to  use  one  volume  of  the  actual  sediment  to  nineteen  volumes  of 
salt  solution. 


THE  TECHNIQUE  OF  SERUM  REACTIONS 


261 


In  all  hemolytic  tests  the  time  element  is  important.  No  hemolysis 
should  be  adjudged  as  incomplete  unless  at  least  one  hour  has  elapsed. 

Isohemolysins  and  Iso-agglutinins. — It  is  often  necessary  to  carry 
out  hemolytic  and  hemagglutinating  tests  on  the  blood  corpuscles  of  one 
human  being  with  the  serum  of  another  in  order  to  determine  the  ad- 
visability of  performing  transfusion.  In  this  case,  the  serum  of  the  re- 
cipient is  mixed  with  a  corpuscle  emulsion  of  the  cells  of  the  donor, 
and  vice  versa.  This  is  conveniently  done  in  small  pipettes  by  the 
method  of  Ottenberg. 

By  the  determination  of  iso-agglutinins  and  isohemolysins  all  human  beings 
can  be  divided  into  four  groups  according  to  Landsteiner  and  others. 


Table  fob 

.  Iso-Agglutinins — Sera 

I 

II 

III 

IV 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

I 

II  - 
m 

IV    ^ 

5 

1 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

2 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

3 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

4 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

i 

5 

+ 

+ 

+ 

+ 

0 

0 

0 

+ 

+ 

0  - 

6 

+ 

+ 

+ 

+ 

0 

0 

0 

+ 

+ 

0    ' 

7 

+ 

+ 

+ 

+ 

0 

0 

0 

+ 

+ 

0 

8 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

0 

0 

0 

9 

+ 

+ 

+ 

4- 

+ 

+ 

+ 

0 

0 

0 

10 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

0 

These  groupings  are  permanent  and  inheritable,  following  MendeFs  laws. 


262  INFECTION  AND  IMMUNITY 

The  Determination  of  Antibodies  in  Sera  by  Complement  Fixation. — 

The  principle  of  complement  fixation,  discovered  by  Bordet  and  Gengou  ^ 
in  1901,  has  been  utilized  both  in  bacteriologicd  investigations,  and  in 
practical  diagnosis  for  the  determination  in  serum  of  the  presence  of 
specific  antibodies.  The  reaction  depends  upon  the  fact  that  when  an 
antigen,  i.e.,  a  substance  capable  of  stimulating  the  formation  of  anti- 
bodies, is  mixed  with  its  inactivated  antiserum,  in  the  presence  of  com- 
plement, the  complement  is  fixed  by  the  combined  immune  body  and 
antigen  can  no  longer  be  found  free  in  the  mixture.  If  such  a  mixture 
is  allowed  to  stand  at  temperature  for  an  hour  or  more,  and  to  it  is 
then  added  an  emulsion  of  red  blood  cells  together  with  inactivated 
hemolytic  serum,  no  hemolysis  will  take  place,  since  there  is  no  free 
complement  to  complete  the  hemolytic  system.  If,  on  the  other  hand, 
the  original  mixture  contains  no  antibody  for  the  antigen  used,  the 
complement  present  is  not  •fixed  and  is  available  for  the  activation  of 
the  hemolytic  serum  later  added. 

The  reaction  thus  depends  upon  the  fact  that  neither  antigen  ^  alone, 
nor  amboceptor  (antibody)  alone,  can  fix  complement,  but  that  this 
fixation  is  carried  out  only  by  the  combination  of  antigen  plus  ambo- 
ceptor. Any  specific  can  be  determined  by  this  method,  provided  the 
homologous  antigen  is  used;  and  vice  versa,  by  the  use  of  a  known  anti- 
body a  suspected  antigen  may  be  determined. 

When  testing  immune  sera  for  antibodies  given  rise  to  in  man  or 
animals  by  microorganisms  which  can  be  cultivated,  either  the  whole 
bacteria  or  extracts  of  the  bacteria  may  be  used  as  an  antigen. 

For  the  diagnosis  of  syphilis  by  this  method,  in  the  so-called  "  Wasser- 
mann  reaction,"  the  antigen  employed  was  originally  obtained  by  the 
extraction  of  sjrphilitic  organs,  in  which  free  syphilitic  antigens,  i.e., 
uncombined  products  of  Spirochaete  pallida,  were  assumed  to  be  present. 

It  has  been  more  recently  shown,  however,  that  the  Wassermann 
reaction  is  not  specific  in  any  sense  of  the  word,  and  that  suitable  anti- 
gens can  be  produced  by  the  alcoholic  extraction  of  lipoids  from  the 
normal  organs  of  many  animals  and  man. 

Bacterial  extracts  for  complement-fixation  can  be  made  in  various 
ways.  The  use  of  thick  salt  solution  suspensions  of  the  cultures  them- 
selves is  not  advisable  because  of  the  anticomplementary  action  of 
such  suspensions.     Good  bacterial  antigens  can  be  produced  by  cen- 


*  Bordet  and  Gengou,  Ann.  de  I'inst.  Pasteur,  xv,  1901. 
2  Bordet  and  Gay,  Ann.  de  I'inst.  Pasteur,  xx,  1906. 


THE  TECHNIQUE  OF  SERUM  REACTIONS  263 

trifugalizing  them  from  salt  solution  suspensions  and  adding  to  about 
20  mgms.,  90  mgms.  of  common  salt,  rubbing  up  with  a  glass  rod  for 
an  hour,  and  then  adding  distilled  water  to  isotonicity.  This  is  the 
method  of  Besredka.  This  method  has  been  used  with  success  by- 
Miller  and  Zinsser  in  the  case  of  tubercle  bacilU  for  complement-fixation 
in  tuberculosis. 

Wassermann  and  Bruck^  prepare  bacterial  antigen  by  emulsifying 
growths  of  about  ten  agar  slant  cultures  in  10  c.c.  of  sterile,  distilled 
water.  This  is  shaken  for  twenty-four  hours  in  a  shaking  apparatus. 
At  the  end  of  this  time  0.5  per  cent  of  carbolic  acid  is  added  and  the 
fluid  cleared  by  centrifugalization. 

The  Wassermann  Test  for  the  Diagnosis  of  Syphilis. ^ — The  sub- 
stances for  the  test  are  the  following: 

I.  The  Antigen. — In  their  original  experiments,  Wassermann  and 
his  collaborators  made  use  of  salt-solution  extracts  of  the  organs  (chiefly 
of  the  spleen)  of  a  syphilitic  fetus.  The  tissue  was  cut  into  small  pieces 
and  to  one  part  by  weight  of  this  substance,  four  parts  of  normal  salt 
solution  and  0.5  per  cent  of  carbolic  acid  were  added.  This  was  shaken 
in  a  shaking  apparatus  for  twenty-four  hours,  and  after  this  the  coarser 
particles  removed  by  centrifugalization.  The  reddish  supernatant 
fluid  was  used  as  the  antigen  and  could  be  preserved  for  a  long  time  in 
dark  bottles  in  the  ice  chest. 

Alcoholic  extracts  of  syphilitic  organs  were  subsequently  used  by  a 
number  of  authors,  syphilitic  liver  being  extracted  for  twenty-four 
hours  with  five  times  the  volume  of  absolute  alcohol.  This  was  filtered 
through  paper  and  the  alcohol  evaporated  in  vacuo  at  a  temperature 
not  above  40°  C.  About  1  gram  of  this  material  was  then  emulsified 
in  100  c.c.  of  salt  solution  to  which  0.5  per  cent  of  carbolic  acid  has 
been  added. 

It  was  soon  found  that  the  Wassermann  antigen  was  a  purely  non- 
specific substance,  and  since  this  discovery  was  made  there  are  few 
laboratories  in  which  syphilitic  organs  are  at  all  used.  It  appears  that 
lipoidal  extracts  from  almost  any  tissue  can  be  employed,  and  that 
fairly  useful  antigens  can  even  be  obtained  with  solutions  of  commer- 
cial lecithin  and  mixtures  of  commercial  lecithin  and  sodium  oleate.  It 
is  apparent,  therefore,  that  in  the  Wassermann  reaction  an  even  sus- 


*  Wassermann  und  Bruck,  Med.  Klinik,  55,  1905,  and  Deut.  med.  Woch.,  xii,  1906. 
2  Wassermann,  Neisser  und  Bruck,  Deut.  med.  Woch.,  xix,  1906;  Wassermann, 
Neisser,  Bruck  und  Schucht,  Zeit.  f.  Hyg.,  Iv,  1906. 


264  INFECTION  AND  IMMUNITY 

pension  of  lipoidal  substances  constitutes  the  antigen,  and  that  the 
complement-fixing  complex  is  made  by  these  antigens  in  combination 
with  some  substance  spoken  of  by  Noguchi  as  ''lipotropic"  in  the 
syphilitic  serum,  which  has  probably  no  relation  to  true  antibody. 
Our  own  work  with  treponema  pallidum  antigen  would  tend  to  con- 
firm this,  as  well  as  the  experience  of  Noguchi,  Craig  and  Nichols, 
Kolmer,  and  6thers,  who  have  found  that  a  pure  treponema  pallidum 
extract  gives  reactions  in  only  a  few  late  tertiary  cases,  running  not 
at  all  parallel  to  the  fixations  obtained  with  non-specific  lipoidal  sub- 
stances. Although  we  are,  at  the  present  writing,  still  in  the  dark 
as  to  whether  the  syphilitic  antigen  depends  for  its  properties  upon 
the  lipoidal  nature  of  the  extracts  or  upon  the  size  and  dispersion  of 
the  particles  present  in  the  extracts,  we  can  still  assert  that  the  test  is 
reliable  and,  with  care  in  execution  and  interpretation,  of  enormous 
value  in  the  diagnosis  of  syphilis.  However,  it  is  necessary  to  recognize 
that  it  is  surely  nofto  specific  antigen-antibody  reaction. 

The  antigens  most  commonly  in  use  today  are  prepared  as  follows: 

1.  Beef  heart  or  guinea-pig  heart  muscle  is  finely  chopped  up  and 
extracted  in  five  times  its  volume  of  absolute  alcohol.  This  mixture 
is  kept  5  to  7  days  in  the  incubator,  being  frequently  shaken.  It  is  then 
filtered  and  titrated.    Human  heart  muscle  may  also  be  used. 

2.  Noguchi's  Acetone  Insoluble  Lipoid  Antigen.  Fresh  spleen  is 
macerated  and  extracted  for  5  to  7  days  in  the  incubator  in  five  times 
its  volume  of  absolute  alcohol,  being  frequently  shaken.  It  is  then  fil- 
tered and  evaporated  to  dryness  with  the  aid  of  a  fan.  The  sticky 
residue  is  taken  up  in  a  small  quantity  of  ether  and  this  ether  solution 
poured  into  four  times  its  volume  of  C.  P.  acetone.  The  floccular  pre- 
cipitate which  forms  is  collected  and  can  be  preserved  under  acetone. 
About  0.2  grams  of  this  paste  is  dissolved  in  5  c.c.  of  ether.  This  is 
shaken  up  with  100  c.c.  of  salt  solution  until  the  ether  is  evapprated. 
The  resulting  antigen  is  titrated. 

3.  Cholesterinized  Antigen.  According  to  the  researches  of  Sachs 
and  Rondoni,  Browning  and  Cruikshank,  and  Walker  and  Swift,  an- 
tigen can  be  made  more  delicate  by  the  addition  of  cholesterin.  Walker 
and  Swift  recommend  that  an  alcoholic  extract  of  human  or  guinea-pig 
heart  be  made  up  to  a  concentration  of  0.4  per  cent  of  cholesterin. 

A  large  number  of  other  antigens  might  be  mentioned,  but  we 
think  that  the  three  mentioned  above  represent  the  most  important, 
and  in  principle  all  of  those  at  present  in  common  use. 

Before  an  antigen  can  be  used  for  the  actual  test,  it  is  necessary  to 


THE  TECHNIQUE  OF  SERUM  REACTIONS  265 

determine  the  quantity  which  will  furnish  a  valid  result.  The  sub- 
stances which  are  used  as  antigens  often  have  the  power,  if  used  in  too 
large  quantity,  of  themselves  binding  complement.  It  is  necessary, 
therefore,  to  determine  the  largest  quantity  of  each  given  antigen  which 
may  be  used  without  exerting  an  anti-complementary  action,  i.e.,  which 
will  not  inhibit  in  the  presence  of  normal  serum  but  which  will  at  the 
same  time  inhibit  hemolysis  when  syphilitic  serum  is  used.  This  is  done 
by  mixing  graded  quantities  of  the  antigen  with  a  constant  quantity 
of  complement  (0.1  c.c.  of  fresh  guinea-pig  serinn),  in  duplicate  sets, 
adding  to  each  tube  of  one  set  0.2  c.c.  of  a  normal  serum,  and  to  the  other 
0.2  c.c.  of  a  known  syphilitic  serum.  These  substances  are  allowed  to 
remain  together  for  one  hour  and  then  red  blood  corpuscles  and  inac- 
tivated hemolytic  serum  are  added.  The  quantity  which  has  given 
complete  inhibition  with  the  syphilitic  serum,  but  absolutely  no  inhibi- 
tion with  normal  serum,  is  the  one  to  be  employed  in  subsequent  re- 
actions. Before  actual  use,  it  is  convenient  to  make  a  dilution  of  antigen 
in  salt  solution  in  such  a  way  that  1  c.c.  shall  contain  the  amount  re- 
quired. Thus  if  0.05  c.c.  is  wanted,  mix  0.5  c.c.  with  9.5  c.c.  salt  solu- 
tion.    Then  1  c.c.  of  this  can  be  added  to  each  tube  in  the  test. 

II.  The  Hemolytic  Serum, — The  hemolytic  amboceptor,  for  the 
reaction,  is  obtained  by  injecting  into  rabbits  the  washed  red  blood 
corpuscles  of  a  sheep.  A  5  per  cent  emulsion  of  the  corpuscles  is  made 
and  of  this  5  c.c,  10  c.c,  15  c.c,  etc.,  are  injected  at  intervals  of  five 
or  six  days.  Three  or  four  graded  injections  of  this  kind  are  usually 
sufficient  to  furnish  a  serum  of  adequate  hemolytic  power.  The  injec- 
tions may  be  made  intraperitoneally  or  intravenously.  About  nine  or 
.ten  days  after  the  last  injection  of  corpuscles,  the  rabbit  is  bled  from  the 
carotid  artery  and  the  serum  obtained  by  pipetting  it  from  the  clot. 

It  is  best  to  have  a  hemolytic  serum  of  high  potency  in  order  that  the 
quantities  used  for  the  reaction  may  be  as  small  as  possible.  This  is 
desirable  because  of  the  fact  that  the  serum  may  contain  small  amounts 
of  precipitins  for  sheep's  serum,  due  to  insufficient  washing  of  the  cor- 
puscles employed  in  the  immunization. 

It  is  necessary  to  carefully  titrate  the  hemolytic  serum.  For  the 
actual  reaction  most  observers  make  use  of  two  hemolytic  units.  A  hem- 
olytic unit  is  the  quantity  of  inactivated  immune  serum  which,  in  the 
presence  of  complement,  suffices  to  cause  complete  hemolysis  in  1  c.c 
of  a  5  per  cent  emulsion  of  washed  blood  corpuscles.  It  is  the  custom 
in  most  laboratories  today  to  halve  all  the  quantities,  using  0.5  c.c  of 
the  suspension  instead  of  1.0  c.c  and  other  ingredients  accordingly. 
18 


266 


INFECTION  AND  IMMUNITY 


Noguchi  ^  has  pointed  out  very  clearly  the  dangers  of  not  delicately 
adjusting  the  quantity  of  amboceptor  used  in  the  reaction.  He  calls 
attention  to  the  experiments  of  Morgenroth  and  Sachs  ^  who  have 
shown  that  the  relationship  between  complement  and  amboceptor 
necessary  for  hemolytic  reactions  is  one  of  inverse  proportions.  In 
their  own  words,  ''in  the  presence  of  larger  quantities  of  amboceptor, 
smaller  quantities  of  complement  suffice, '^  and  vice  versa.  Noguchi, 
in  his  work,  has  found  that,  while  in  the  presence  of  one  unit  of  ambo- 
ceptor, 0.1  c.c.  of  guinea-pig's  complement  is  required  to  produce 
hemolysis,  by  using  four,  eight,  and  twenty  units  of  amboceptor,  com- 
plete hemolysis  is  obtainable  with  one-third,  one-fifth,  and  one-tenth 
of  the  0.1  c.c.  of  complement,  respectively.  For  this  reason  an  excess 
of  amboceptor  might  result  in  complete  hemolysis  in  a  test,  if  a  small 
fraction  of  the  complement  were  left  unfixed  by  the  syphilitic  antibody. 
Another  result  of  an  excess  of  amboceptor  would  consist  in  a -partial 
dissociatijon  of  the  complement  from  its  combination  with  the  antigen- 
antibody  compound.  As  Noguchi  puts  it,  "a  quantity  of  syphilitic 
antibody  just  sufficient  to  fix  0.1  c.c.  of  the  complement  against  two 
units  of  the  amboceptor  is  no  longer  efficient  in  holding  back  the  com- 
plement from  partial  liberation  against  the  influence  exerted  by  more 
than  four  units  of  the  amboceptor." 

From  these  considerations  it  follows  that  the  serum  from  rabbits 
immunized  against  sheep  corpuscles  must,  in  each  case,  be  titrated  in 
order  to  determine  the  hemolytic  unit.  For  this  purpose  a  number  of 
mixtures  are  made  in  test  tubes,  containing  each  0.1  c.c.  of  complement 
(fresh  guinea-pig  serum),  1  c.c.  of  a  5  per  cent  emulsion  of  sheep's  cor- 
puscles, and  diminishing  quantities  of  the  inactivated  hemolytic  serum, 
thus: 

=  complete  hemolysis 
=  complete  hemolysis 
=  complete  hemolysis 
=  complete  hemolysis 
=  complete  hemolysis 
,0009  c.c.  =  partial  hemolysis 
.0005  c.Q.  =  no  hemolysis 
.0003  c.c.  =  no  hemolysis.  ^ 


» 

1  c.c. 

.1  c.c.  of      " 

of  5  per 
cent 
emul- 
sion 
sheep's 
corpus- 

Inac- 

complement 

tivated 

fresh 

guinea-pig 

serum. 

-  +  - 

'  +  - 

hemo-   * 
lytic 
serum. 

cles. 

.01 

c.c. 

.009 

c.c. 

.005 

c.c. 

.003 

c.c. 

.001 

c.c. 

1  Noguchi,  Proc.  Soc.  for  Exper.  Biol,  and  Med.,  VI,  3,  1909. 

2  Morgenroth  und  Sachs,  in  Ehrlich's  "Gesammelte  Arbeiten,"  etc.,  Berhn,  1904. 

3  In  each  tube  the  volume  of  the  mixture  should  be  made  up  to  5  c.c.  with  0.85 
per  cent  salt  solution. 


THE  TECHNIQUE  OF  SERUM  REACTIONS  267 

In  the  given  case,  0.001  c.c.  of  the  serum  represents  one  unit,  and 
0.002  c.c,  two  units,  is  the  quantity  to  be  used  for  each  test. 

III.  The  Complement. — The  complement  used  in  Wassermann  re- 
action is  fresh  guinea-pig  serum.  This  may  be  obtained  in  one  of  the 
following  ways :  A  guinea-pig  may  be  killed  by  an  incision  in  the  throat 
and  the  blood  allowed  to  flow  into  a  large  Petri  dish.  This  is  set  away 
in  the  ice  chest  until  clear  beads  of  serum  have  formed  upon  the  sur- 
face, and  these  are  then  carefully  removed  with  a  pipette. 

It  is  more  economical  to  puncture  the  heart  of  large  guinea-pigs 
with  a  needle  attached  to  a  syringe  and  withdraw  5  or  6  c.c.  of  blood 
without  Idlling  the  animal.  This  can  be  transferred  to  a  centrifuge 
tube  and  the  serum  obtained  by  centrifugation  after  clotting.  Serum 
used  as  complement  in  the  Wassermann  reaction  must  be  titrated  each 
day  before  reactions  are  done.  This  is,  done  by  putting  into  a  series  of 
tubes  1.0  c.c.  (or  if  half  quantities  are  used,  as  with  us,  0.5  c.c.)  of  the 
cell  suspension  sensitized  with  2  units  of  amboceptor,  and  adding  to 
these  tubes  varying  quantities  of  guinea-pig  serum.  The  guinea-pig 
serum  is  best  diluted  1:10  in  salt  solution,  and  quantities  ranging  from 
0.05  to  0.35  c.c.  are  added  to  the  tubes.  The  unit  is  the  amount  in  the 
tube  which  shows  complete  hemolysis  at  the  end  of  an  hour.  The  re- 
actions are  usually  complete  in  about  30  minutes.  Two  units  of  the  com- 
plement are  used  in  the  ordinary  test.  The  titration  of  the  complement 
is  one  of  the  most  important  steps  in  accurate  work. 

IV.  The  Sheep  Corpuscles. — The  sheep  corpuscles  for  the  actual  re- 
action are  obtained  by  receiving  the  blood  in  a  small  flask  containing 
a  sterile  solution  of  a  0.5  per  cent  sodium  citrate  and  0.85  per  cent 
sodium  chloride,  or  into  one  containing  glass  beads  or  short  pieces  of 
glass  tubing.  In  the  former  case,  the  citrate  solution  prevents  clotting 
and  the  corpuscles  may  be  washed  free  from  the  citrate  solution  and 
emulsified  in  salt  solution  before  use  in  the  test.  In  the  latter  case,  it 
is  necessary  to  shake  the  blood  in  the  flask  immediately  after  taking, 
and  to  continue  the  shaking  motion  for  about  ten  minutes.  The  cor- 
puscles are  washed  free  from  serum  by  at  least  3  washings  in  salt  solu- 
tion. A  5  per  cent  suspension  of  the  corpuscles  is  employed  for  the  test, 
made  by  measuring  the  bulk  of  centrifugalized  corpuscles  and  adding 
nineteen  parts  of  sterile  salt  solution. 

V.  The  Serum  to  he  Tested  for  Syphilitic  Antibody. — The  serum  of 
the  patient  is  best  obtained  in  the  same  way  that  blood  is  obtained  for 
blood  cultures.  After  surgical  precautions,  a  needle  is  plunged  into  the 
median  basilic  vein  and  3  or  4  c.c.  of  blood  are  removed.    Before  use 


268  INFECTION  AND  IMMUNITY    ' 

\ 
for  the  test,  the  patient's  serum  must  be  inactivated  by  heating  in  a 
water  bath  to  56°  C.  for  twenty  minutes  to  half  an  hour. 

The  Test. — The  actual  test  for  antibody  in  a  suspected  serum  is 
carried  out  in  the  following  way:  In  a  test-tube  of  suitable  size,  2  units 
of  the  complement,  0.2  c.c.  of  the  inactivated  suspected  serum,  and  the 
antigen,  in  quantity  determined  by  titration,  are  mixed,  and  the  total 
volume  brought  up  to  3  c.c.  with  normal  salt  solution.  This  mixture  is 
thoroughly  shaken,  and  placed  for  one  hour  in  a  water  bath  or  in  the 
incubator  at  37.5°  C.  Recently  it  has  been  found  that  more  delicate 
results  are  obtained  when  the  fixation  is  allowed  to  take  place  in  the 
refrigerator  for  three  or  four  hours — the  so-called  *' ice-box  method." 
At  the  end  of  this  preliminary  incubation  there  is  added  1  c.c.  of  a  5 
per  cent  emulsion  of  sheep's  corpuscles,  and  two  units  of  hemolytic 
amboceptor,  determined  by  a  titration  of  the  inactivated  hemolytic 
rabbit  serum,  as  described  above.  This  inixture  is  again  placed  at  37.5° 
C.  for  one  to  two  hours.  If  the  antibody  is  present  in  the  suspected 
serum,  no  hemolysis  takes  place.    If  absent,  hemolysis  is  complete. 

In  our  own  work  all  tests  are  done  in  half  the  quantities  of  the 
original  Wassermann.  Hence  only  0.1  c.c.  of  the  patient's  serum,  and 
the  antigen  and  complement  as  determined  in  titrations  with  0.5  c.c. 
of  the  cells  are  mixed  in  a  total  volume  of  1.5  c.c.  At  the  end  of  the 
preliminary  incubation,  0.5  c.c.  of  cells  previously  sensitized  with  2 
units  of  amboceptor  are  added. 

No  test  is  of  use  unless  suitable  controls  are  made.  The  controls 
set  up  should  be  as  follows: 

Control  1.  For  each  serum  tested  the  mixture  described  above, 
omitting  antigen. 

Controls  2  and  3.  The  mixture  made  as  in  the  test  but  with  known 
syphilitic  serum  (2)  with  and  (3)  without  antigen. 

Controls  4  and  5.  The  mixture  made  as  in  the  test,  but  with  normal 
serum  (4)  with  and  (5)  without  antigen. 

Controls  6  and  7.  The  hemolytic  system,  complement,  blood  cells 
and  amboceptor,  set  up  in  order  to  show  that  the  system  is  in  working 
order  (6)  with  and  (7)  without  antigen.  It  is  convenient  to  set  the 
tubes  in  two  rows  in  a  rack,  the  front  row  containing  antigen,  the  back 
row  containing  the  same  mixture  without  antigen. 

In  a  positive  test,  the  test  itself,  and  Control  2,  alone,  should  show 
inhibited  hemolysis.  The  other  tubes  should  show  complete  solution 
of  the  hemoglobin.     (See  scheme,  p.  259.) 

Modifications  of  the  Wassermaim  Test. — Since  the  original  for- 


THE  TECHNIQUE  OF  SERUM  REACTIONS  269 

mulation  of  the  Wassermann  reaction  a  great  many  modifications  have 
been  suggested  by  various  workers,  some  of  them  being  radical  changes 
involving  the  altering  of  the  hemolytic  system;  others,  however,  merely 
adding  precautions  here  and  there  to  increase  the  delicacy  of  the  reac- 
tion. The  literature  on  this  subject  is  too  voluminous  to  be  com- 
pletely covered.  We, indicate,  therefore,  some  of  the  most  important 
changes  from  the  original  that  have  been  found  valuable,  and  give  in 
greater  detail  the  methods  as  -at  present  in  use  in  our  own  laboratory. 

Bauer  has  called  attention  that  human  serum  contains  a  certain  amount  of 
natural- hemolysin  for  sheep  corpuscles.  In  his  original  modification,  therefore,  he 
does  not  use  hemolytic  rabbit  serum  as  amboceptor.  His  modification  as  a  whole 
cannot  be  accepted  for  general  use  because  human  sera  do  not  contain  a  uniform 
amount  of  hemolysin  for  sheep  cells,  and  some  contain  none  whatever.  However, 
the  presence  of  natural  amboceptor,  so-called,  in  human  sera  is  taken  account  of 
by  many  workers,  and  it  is  important  to  recognize  this,  since  naturally  it  adds  to 
the  amboceptor  added  with  sensitized  cells  -and  leads  to  a  lack  of  uniformity  in 
the  dosage  of  amboceptor  in  individual  tubes  if  included. 

Noguchi  has  worked  out  a  test  in  which  the  difficulties  presented  by  the  pres- 
ence of  normal  sheep  amboceptor  are  eliminated,  in  that  he  uses  an  antihuman 
hemolytic  serum  and  human  cells  as  the  hemolytic  system.  It  enables  him  also  to 
use  the  cells  of  the  patient  or  of  any  other  human  being,  thus  eliminating  the  neces- 
sity of  getting  fresh  sheep  cells.    His  tests  are  set  up  as  follows: 

Tube  1.  1  drop  patient's  serum  +  complement  (0.1  c.c.  of  40  per  cent  guinea-pig  serum)  +  antigen- 
Tube  2.  1  drop  patient's  serum  4- complement.     (No  antigen.) 
Tube  3.  1  drop  known  syphilitic  serum  +  complement  +  antigen. 
Tube  4.  1  drop  known  syphilitic  serum  +  complement.     (No  antigen.) 
Tube  5.  1  drop  known  normal  serum  +  cojTiplement  + antigen. 
Tube  6.  1  drop  known  normal  serum  +  complement.      (No  antigen.) 
Tube  7.  Complement  alone  (for  hemolytic  system  control). 

To  each  tube  then  add  1.0  c.c.  of  the  one  per  cent  emulsion  of  human  corpuscles. 
Shake  mixtures  thoroughly  and  incubate  or  place  in  water  bath  at  38-40°  C.  for  one 
hour.  Then  add  to  each  tube  2  units  of  antihuman  amboceptor  (serum  of  rabbit 
immunized  with  human  cells)  and  replace  in  water  bath  for  one  hour.  At  the  end 
of  this  time  in  a  positive  test  there  wiU  be  no  hemolysis  in  Tubes  1  and  3  while  all 
the  other  tubes  will  show  hemolysis. 

The  method  of  Noguchi  is  still  used  by  a  few  investigators,  but  is 
not  at  present  in  common  use,  though  we  have  no  doubt  that  if  sys- 
tematically followed  it  would  develop  as  quite  satisfactory. 

The  tests  are  done  in  our  own  laboratory  with  the  original  sheep 
cell — antisheep  serum  hemolytic  system.  They  are  done  in  half  quan- 
tities, titrations  being  made  with  0.5  c.c.  of  a  5  per  cent  emulsion  of 
washed  sheep  cells.  Each  day  the  complement  (fresh  guinea-pig  diluted 
1  :  10)  is  titrated  with  cells  sensitized  with  2  units  of  stock  amboceptor. 
Fresh  amboceptors  are  titrated  from  time  to.  time  so  that  a  reasonable 
constancy  is  obtained.  The  hemolytic  system  is  kept  as  constant  as 
possible  from  day  to  day.  The  unit  (minimal  hemolytic  amount)  of 
a  new  specimen  of  amboceptor  is  determined  by  titrating  it  on  a  num- 
ber of  successive  days  with  0.5  c.c.  of  5  per  cent  cells  and  0.5  c.c.  of 
10  per  cent  guinea-pig  sera,  readings  being  made  at  the  end  of  a  half 


270 


THE  TECHNIQUE  OF  SERUM  REACTIONS 


hour.  When  complement  is  then  subsequently  titrated  with  2  such 
amboceptor  units  and  0.5  c.c.  of  5  per  cent  red  cells,  it  is  usually  found 
that  the  minimal  hemolytic  dose  of  complement  lies  between  0.2  and 
0.25  c.c,  and  in  the  actual  tests  twice  this  minimal  hemolytic  dose  of 
complement  is  used  with  the  2  units  of  amboceptor.  The  daily  titra- 
tion of  complement  frequently  shows  marked  variations  even  though 

SCHEME  FOR   WASSERMANN  TEST. 

ADAPTED    TO    ORIGINAL    WASSERMANN    SYSTEM    AFTER    SCHEME    OF    NOGUCHI. 


Test  with  Unknown 
Serum. 

Test  with  Known 

Positive  Syphilic 

Serum. 

Test  with  Known 

Negative  Normal 

Serum. 

Test  without  Serum 

to  Control  Efficiency 

of  Hemolytic 

System. 

£H 

Serum  .2  c.c. 

d 

6 

Serum  ..2  c.c. 

Serum  .2  c.c. 

■ 

^1 

.+' 

CO 

+ 

+. 

^1 

Q   Complement 

s 

0   Complement 

Q   Complement 

0    Complement 

g    = 

.1  c.c. 

1 

.1  c.c. 

.1  c.c. 

.1  c.c. 

^9 

+ 

> 

+ 

+ 

+ 

g 

Salt  sol.  3.  c.c. 

3 

o 

Salt  sol.  3.  c.c. 

Salt  sol.  3.  c.c. 

Salt  sol.  3.  c.c. 

2. 

A 

4, 

6. 

8. 

Serum  .2  c.c. 

Serum  .2  c.c. 

Serum  .2  c.c. 

+ 

6 

+ 

+ 

o  <o 

Q  Complement 

CO 

0    Complement 

0    Complement 

0 .  Complement 

^.^ 

.1  c.c. 

.1  c.c. 

.1  c.c. 

.1  c.c 

%< 

4- 

+ 

+ 

+ 

-1 

Antigen 

(required  amount 

Antigen 

Antigen 

Antigen 

in  1  c.c.  salt  sol.). 

+ 

I 

+ 

+ 

+ 

Salt  sol.  2.  c.c. 

Salt  sol.  2.  c.c. 

Salt  sol.  2.  c.c. 

Salt.  sol.  2.  c.c. 

1. 

1  .3. 

5. 

7. 

O  ==  test  tube. 

Place  in  water  bath  at  40°  C.  for  one  hour,  then  add  to  all  tubes  red  blood  cells  and 
amboceptor.  These  are  previously  mixed  so  that  2  c.c.  contains  the  equivalents  of 
1  c.c.  of  a  5  per  cent  emulsion  of  sheep  corpuscles  and  2  units  of  amboceptor.  Again 
expose  to  40°  C.  If  the  serum  tested  is  positive,  tubes  1  and  3  should  show  no 
hemolysis,  all  the  other  tubes  showing  complete  hemolysis  in  one  hour. 

the  complement  is  obtained  from  a  number  of  guinea-pigs.  By  always 
using  twice  the  amount  which  just  lakes  with  2  units  of  amboceptor, 
one  keeps  the  actual  amount  of  complement  as  nearly  as  possible 
constant. 

Obviously  one  can  titrate  the  strength  of  the  reaction  by  varying 
the  amount  of  any  one  of  the  three  ingredients  which  primarily  enter 


THE  TECHNIQUE  OF  SERUM  REACTIONS  271 

into  it — the  antigen,  the  patient's  serum,  or  the  complement,  and  all 
three  of  these  systems  have  been  proposed  and  used  with  success  by- 
different  workers.  The  method  which  is  most  commonly  used  is  that 
of-  Citron  and  consists  of  using  diminishing  quantities  of  both  antigen 
and  patient's  serum.  Citron  in  addition  to  the  main  tube  uses  one 
additional  tube  containing  one-half  the  amount  of  patient's  serum 
and  one-half  the  amount  of  antigen  used  in  the  main  tube,  and  he  ex- 
presses his  results  as  follows: 

If  both  tubes  show  complete  inhibition +  +  +  + 

If  the  main  tube  shows  complete  inhibition  and  the  half  dose 

tube  almost  complete +  +  + 

If  the  main  tube  shows  complete  inhibition  and  the  second 

tube  faint  inhibition +  -f- 

•If  the  main  tube  shows  incomplete  inhibition  and  the  second 

tube  none  or  little -j- 

If  the  main  tube  shows  very  faint  inhibition  and  the  second 

tube  shows  none ± 

"  +  +  ++,"  "  +  ++,"  and  "  +  +  "  are  regarded  as  conclusive  results; 
"-h"  as  a  probable  positive;  "  ±  "  as  suspicious  merely. 

The  Determination  of  Antigen  by  Complement  Fixation. — The  prin- 
ciples underlying  the  preceding  tests  for  the  determination  of  sus- 
pected antibodies  may  be  equally  applied  to  the  determination  of 
suspected  antigen.  In  the  former  case  it  was  necessary  to  bring  the 
serum  to  be  tested  into  contact  with  the  antigen  specific  for  the  suspected 
antibody,  in  the  presence  of  complement,  and  at  a  suitable  tempera- 
ture. At  the  end  of  an  hour  the  mixture  was  tested  for  free  comple- 
ment by  the  addition  of  hemolytic  amboceptor  and  red  blood  cells. 
In  testing  for  antigen,  the  procedure  is  reversed,  in  that  the  serum  or 
other  substance  (bacterial  extract)  to  be  tested  is  brought  into  contact 
with  an  antibody  specific  for  the  antigen,  in  the  presence  of  complement ; 
and  at  the  end  of  an  hour  at  suitable  temperature,  free  complement 
is  again  determined  by  hemolytic  reaction  as  before. 

When  dealing  with  bacterial  antigen,  it  is  necessary,  therefore,  to 
prepare  a  highly  potent  immune  serum  against  the  bacteria  which 
contain  the  specific  antigen  which  is  sought. 

Thus  in  testing  for  typhoid-bacillus  antigen  in  the  serum  of  a  patient, 
the  substances  required  are  as  follows: 

1.  Complement:  obtained  from  fresh  guinea-pig  serum.  It  is  best 
to  titrate  the  complement  when  possible,  using  for  the  test  double  the 


272  INFECTION  AND  IMMUNITY 

quantity  necessary  to  produce  complete  hemolysis  of  1  c.c.  of  a  five 
per  cent  emulsion  of  blood  cells,  in  the  presence  of  two  units  of  ambo- 
ceptor. 

2.  Hemolytic  amboceptor:  rabbit  serum  hemolytic  for  sheep  cor- 
puscles.    Inactivated  and  titrated  as  for  Wassermann  test. 

3.  A  five-per-cent  emulsion  of  sheep  corpuscles  in  salt  solution,  pre- 
pared as  for  Wassermann  test. 

4.  A  highly  potent  typhoid  antiserum  obtained  from  an  immunized 
rabbit.  In  this  case  the  smallest  quantity  of  the  immune  serum  which 
will  cause  the  fixation  of  complement  in  the  presence  of  an  emulsion  or 
extract  of  known  typhoid  bacilli  is  determined  by  experiment. 

It  is  best  to  rub  up  the  centrifugalized  bacteria  with  dry  salt,  adding 
distilled  water  to  isotonicity.  When  the  extract  is  made,  its  anticom- 
plementary dose  is  determined  and  the  minimum  quantity  which  in  the  • 
presence  of  known  antityphoid  serum  will  fix  complement.  These  pre- 
liminary titrations  are  analogous  to  those  described  as  preliminary  to 
the  Wassermann  test.  When  these  quantities  have  been  determined, 
an  amount  of  the  bacillary  extract  (about  1-3  or  1-4  of  the  anticom- 
plementary dose)  is  chosen  for  the  actual  tests.  It  is  well,  also,  in  such 
tests  to  determine  the  amount  which  will  fix  complement  in  the  pres- 
ence of  a  known  normal  serum,  since  occasional  presence  of  antibodies 
against  some  bacteria  in  normal  serum  may  otherwise  confuse  the  test. 
The  quantities  of  antigen  and  complement  must  then  be  chosen  for  the 
test  in  such  proportions  that  no  fixation  will  occur  with  normal  serum. 

5.  Serum  from  the  patient,  inactivated  at  56°  C.  for  twenty 
minutes. 

In  the  actual  test  a  series  of  tubes  are  prepared  each  of  which  con- 
tains: 

1.  Complement,  the  determined  quantity. 

2.  Antiserum,  the  determined  quantity. 

3.  Diminishing  quantities  of  the  inactivated  serum  to  be  tested  for 
antigen  beginning  with  1  c.c. 

Salt  solution  is  added  for  dilution  to  3  c.c 

These  substances  are  left  together  at  37°  to  40°  C.  for  one  hour  and 
then  the  required  quantities  of  amboceptor  and  red  cells  are  added. 
The  reaction  is  controlled  by  tubes  containing  the  same  ingredients 
without  the  tjrphoid  antiserum.  In  a  positive  test  there  will  be  no 
hemolysis  in  the  tubes  containing  the  patient's  serum. 


tf  HE  TECHNIQUE  OF  SERUM  REACTIONS  273 

Protein  Differentiation  by  Complement  Fixation. — That  the  technique 
of  complement  fixation  was  applicable  to  the  determination  of  specific 
proteid  antigen — such  as  human  or  animal  blood — was  shown  by  Gen- 
gou  1  in  1902.  The  principles  worked  out  by  him  have  been  pi;actically 
applied  by  Neisser  and  Sachs  ^  and  others  to  the  forensic  differentiation 
of  animal  proteids  and  these  tests  are  said  to  be  more  delicate  and 
reliable  than  precipitation  tests  made  for  the  same  purpose. 
The  substances  necessary  for  the  reaction  are  as  follows: 

1.  Complement,  titrated  as  above. 

2.  Hemolytic  amboceptor  as  above. 

3.  A  five-per-cent  emulsion  of  sheep, corpuscles  as  above. 

4.  Specific  antiserum. 

This  is  obtained  from  a  rabbit  immunized  with  the  proteid  for  which 
the  test  is  to  be  made;  viz.;  human  or  animal  blood  serum.  This  must 
be  titrated.  In  order  to  do  this,  diminishing  quantities  of  the  antiserum 
are  mixed  in  a  series  of  tubes  with  the  determined  quantity  of  comple- 
ment, and  the  antigen  which  is  to  be  tested  for,  i.e.,  the  homologous 
serum  with  whiich  the  antiserum  has  been  produced.  Since  the  test 
should  be  sufficiently  delicate  to  determine  0.0001  c.c.  of  the  antigen, 
this  quantity  is  added  to  each  tube.  The  actual  titration  is  as 
follows  :^^ 

1.  Antiserum,  undiluted       .1  +  homologous  serum  .0001  +  complement. 

2.  Antiserum  diluted  by  10.75+  homologous  serum  .0001  +  complement. 

3.  Antiserum  diluted  by  10.75+  homologous  serum  .0001  +  complement. 

4.  Antiserum  diluted  by  10.3  +  homologous  serum  .0001  +  complement. 

etc.,  down  to  .1 

These  tubes  are  incubated  for  one  hour  and  hemolytic  amboceptor 
and  red  blood  cells  are  added.  The  smallest  quantity  of  antiserum 
which  has  completely  inhibited  hemolysis  is  the  "unit,"  and  one  and  a 
half  to  two  times  this  quantity  is  used  for  the  test. 

5.  A  solution  of  the  blood  spot  or  other  material  to  be  tested  pre- 
pared as  for  precipitin  test.     (See  page  254.) 

For  the  actual  test  the  following  mixtures  are  made  in  a  series  of 
tubes,  each  of  which  contains: 


1  Gengou,  Ann.  de  V  Inst.  Pasteur,  1902. 

2  Neisser  und  Sachs,  Berl.  klin.  Woch.,  1905  and  1906,    See  also  Citron,  in  Kraua 
and  Levaditi  "Handbuch,"  etc. 

3  Citron,  loo.  cit. 


274  INFECTION  AND  IMMUNITY  • 

.     omp  1  qyg^jj^j^y  detemiined  by  titration. 

2.  Antiserum      J 

3.  Diminishing  quantities  of  the  substance  in  which  the  antigen  is  suspected, 
ranging  from  0.1  c.c.  downward  to  0.0001  c.c. 

Salt  solution  is  added  as  a  diluent  up  to  3  c.c.  and  the  tubes  are 
placed  in  the  incubator  or  water-bath  at  37.5°  to  40°  C.  At.  the  end 
of  this  time  red  blood  cells  and  amboceptor  are  added  as  before. 

The  tubes  are  controlled  by  a  series  containing  all  the  above  ingredi- 
ents except  the  antiserum. 


CHAPTER  XVII 


PHAGOCYTOSIS 


The  studies  on  immunity  which  we  have  outlined  in  the  preceding 
sections  have  dealt  entirely  with  the  phenomena  occurring  in  the  re- 
action between  bacteria  or  bacterial  products  and  the  body  fluids.  These 
studies,  we  have  seen,  have  formed  the  basis  of  a  theoretical  conception 
of  immunity  formulated  chiefly  by  the  German  school  of  bacteriologists 
under  the  leadership  of  EhrHch,  Pfeiffer,  Kruse,  and  others.  Parallel 
with  these  developments,  however,  investigations  on  immunity  have 
been  carried  on  which  have  brought  to  Hght  many  important  facts  con- 
cerning the  participation  of  the  cellular  elements  of  the  body  in  its 
resistance  to  infectious  germs. 

The  inspiration  for  this  work  and  the  greater  part  of  the  theoretical 
/considerations  which  have  been  based  upon  it  have  emanated  from 
Metchnikoff  ^  and  his  numerous  pupils  at  the  Pasteur  Institute  in  Paris. 
The  phenomenon  which  these  observers  have  studied  in  great  detail 
and  upon  the  occurrence  of  which  they  have  based  their  conceptions  of 
immunity,  is  known  as  phagocytosis. 

It  is  well  known  that  among  the  lowest  unicellular  animals  the 
nutritive  process  consists  in  the  ingestion  of  minute  particles  of  organic 
matter  by  the  cell.  The  rhizopods,  which  may  be  found  and  studied 
in  water  from  stagnant  pools  or  infusions,  when  observed  under  the 
microscope,  may  be  seen  to  send  out  short  protoplasmic  processes,  the 
pseudopodia,  by  means  of  which  they  gradually  flow  about  any  foreign 
particle  with  which  they  come  in  contact.  If  the  ingested  particle  is 
of  an  inorganic  nature  and  indigestible,  it  will  be  again  extruded  after  a 
varying  period.  If,  however,  the  ingested  substance  is  of  a  nature  which 
can  be  utilized  in  the  nutrition  of  the  protozoon,  it  is  rapidly  surrounded 
by  a  small  vacuole  within  which  it  is  gradually  dissolved  and  becomes  a 
part  of  the  cellular  protoplasm.  This  digestion  within  the  unicellular 
organism  is  probably  due  to  a  proteolytic  enzyme  ^  which  acts  in  the 

Metchnikoff,  "  L'lmmunit^  dans  les  maladies  infectueuses." 
*  Mouton,  Ann.  de  I'inst.  Pasteur,  xvi,  1902. 
275 


276  INFECTION  AND  IMMUNITY 

presence  of  a  weakly  alkaline  reaction.  This  has  been  shown  by  the 
actual  extraction,  from  amebae,  of  a  trypsin-like  ferment. 

As  we  proceed  higher  in  the  scale  of  the  animal  kingdom,  we  find  that 
this  power  of  intracellular  digestion,  while  not  uniformly  an  attribute  of 
all  the  body  cells,  is  still  well  developed  and  a  necessary  physiological 
function  of  certain  cells  which  have  retained  primitive  characters.  In 
animals  like  the  coelenterata,  in  which  there  are  two  cell  layers,  an 
entoderm  and  an  ectoderm,  the  ectodermal  cells  have  lost  the  power  of 
intracellular  digestion,  while  the  entodermal  cells  are  still  able  to  ingest 
and  digest  suitable  foreign  particles.  It  is  only  as  we  proceed  to  animals 
of  a  much  higher  organization  that  the  function  of  cell  ingestion  of  crude 
food  is  entirely  removed  from  the  process  of  general  nutrition.  Never- 
theless, in  these  animals  also,  the  actual  cell  ingestion  of  foreign  particles 
occurs,  but  it  is  now  limited  entirely  to  a  definite  group  of  cells.  In  the 
higher  animals  and  in  man,  this  function  of  phagocytosis  is  limited 
to  the  white  blood  cells  of  the  circulation,  or  leucocytes,  to  certain  large 
endotheHal  cells  lining  the  serous  cavities  and  blood-vessels,  and  to  cells 
of  a  rather  obscure  origin  which  contribute  to  the  formation  of  giant 
cells  within  the  tissues.  A  convenient  division  of  these  phagocytic  cells 
is  that  into  ''wandering  cells"  and  "fixed  cells.''  The  wandering  cells 
are  the  polymorphonuclear  leucocytes,  called  "  microphages "  by 
Metchnikoff,  and  certain  large  mononuclear  elements  or  "macrophages." 
Fixed  cells,  also  called  macrophages  by  Metchnikoff  and  possessing  the 
power  of  ameboid  motion,  include  the  cells  lining  the  serous  cavities, 
and  the  blood  and  lymph  spaces.  The  small  lymphocytes,  so  far  as  we 
know,  have  no  phagocytic  functions. 

In  studying  the  cellular  activities  which  come  into  play  whenever 
foreign  material  of  any  description  gains  entrance  into  the  animal  body, 
a  definite  reaction  on  the  part  of  the  phagocytic  cells  may  be  observed. 
When  we  inject  into  the  peritoneal  cavity  of  a  guinea-pig  a  small  quan- 
tity of  nutrient  broth,  and  examine  the  exudate  within  the  cavity  from 
time  to  time,  we  can  observe  at  first  a  diminution  from  the  normal  of 
the  cells  present  in  the  peritoneal  fluid.  This  may  be  due  either  to  an 
injury  of  the  leucocytes  by  the  injected  substance,  or  to  an  actual  repel- 
lent mfluence  which  the  injected  foreign  material  exerts  upon  the  wan- 
dering cells .^  Very  soon  after  this,  however,  the  exudate  becomes  ex- 
tremely rich  in  leucocytes,  chiefly  of  the  polymorphonuclear  i^ariety ,  the 
maximum  of  the  reaction  being  reached  about  eighteen  to  twenty-four 

1  Pierrallini,  Ann.  de  Tinst.  Pasteur,  1897. 


PHAGOCYTOSIS  277 

hours  after  the  injection.  After  this,  there  is  a  gradual  diminution  in 
the  leucocytic  elements  until  the  fluid  in  the  peritoneal  cavity  again 
reaches  its  normal  condition.  It  is  plain,  therefore,  that  the  presence 
of  the  foreign  material  in  the  peritoneal  cavity  has,  after  a  primary 
repellent  action  upon  the  phagocytes,  attracted  them  in  large  numbers 
to  the  site  of  the  foreign  substance.  Such  repelling  or  attracting  in- 
fluences upon  the  leucocytes  are  spoken  of  as  negative  or  positive 
chemotaxis.     The  reasons  for  chemotaxis  are  not  well  understood.     In 

M       i_iiMii    TT '•  •-»'•"  .... 

the  case  of  bacteria,  which  chiefly  interest  us  in  the  present  connection, 
chemotactic  attraction  or  repulsion  is  intimately  dependent  upon  the 
nature  of  the  microorganism,  and  very  probably  has  a  definite  relation- 
ship to  its  virulence.  Whether  or  not  the  principles  of  chemotaxis  may 
serve  to  explain  the  hypo-  and  hyper-leucocytoses,  observed  and  diag- 
nostically  utilized  in  clinical  medicine,  is  by  no  means  positive.  It  is 
likely,  however,  that  the  two  phenomena  are  closely  associated.  Leva- 
diti  ^  believed  that  he  obtained  some  evidence  that  negative  chemotaxis 
may  take  place  within  the  blood-vessels  when  he  noticed  that  the  intra- 
venous injection  of  cholera  spirilla  into  immunized  guinea-pigs  resulted 
in  an  immediate  disappearance  of  leucocytes  from  the  circulating  blood, 
and  their  accumulation  in  the  internal  organs.  On  the  other  hand,  this 
may  possibly  be  more  logically  explained  by  a  concentration  of  both 
bacteria  and  leucocytes  in  the  capillary  system  of  such  an  organ  as  the 
liver,  as  it  is  known  that  injected  bacteria  rapidly  disappear  from  the 
general  circulation,  but  may  be  demonstrated  in  the  various  organs  for 
some  time  after  injection. 

We  have  seen,  therefore,  that  the  invasion  of  the  animal  body  by 
foreign  material,  living  or  dead,  is  followed  by  a  prompt  response  on  the 
part  of  the  phagocytic  cells.  In  the  -  case  of  bacteria,  when  these  are 
deposited  in  the  subcutaneous  areolar  tissues,  the  inflammatory  reaction 
which  follows  brings  with  it  an  emigration  of  microphages  (polynuclear 
leucocytes)  from  the  blood-vessels — and  these  are  the  so-called  pus 
cells.  When  the  injection  of  bacteria  is  intraperitoneal,  after  a  primary 
diminution,  there  is  an  increase  of  leucocytes  in  the  peritoneal  cavity 
which  soon  results  in  the  formation  of  a  copious  turbid  exudate.  If  the 
pus  of  an  abscess  or  the  exudate  from  an  infected  peritoneum  is  ex- 
amined microscopically,  it  will  be  seen  that  many  of  the  microphages 
have  taken  bacteria  into  their  cytoplasm.  That  fully  virulent  living 
bacteria  can  be  so  taken  up  has  been  variously  proven.     The  phago- 

^Levaditi,  Presse  med.,  1900. 


278  INFECTION   AND   IMMUNITY 

cjrtosis  is,  therefore,  not  simply  a  removal  of  the  dead  bodies  of  bacteria 
previously  killed  by  the  body-fluids,  but  represents  an  actual  attack  upon 
living  and  fully  virulent  microorganisms.  That  the  ingested  bacteria  are 
often  alive  after  ingestion  is  proved  by  the  fact  that  the  injection  of  exu- 
date containing,  so  far  as  can  be  determined,  only  intracellular  bacteria, 
has,* in  several  instances,  been  found  to  give  rise  to  infection. 

Afte^:.the  bacteria  have  remained  for  some  time  within  the  cytoplasm 
of  the  leucocyte,  vacuoles  may  be  seen  to  form  about  them,  similar  to 
those  mentioned  in  discussing  the  digestive  processes  of  amebse.  If  the 
preparations  are,  at  this  stage  or  later,  stained  with  a  one-per-cent 
solution  of  neutral  red,  it  will  be  found  that  the  bacteria,  colorless  under 
normal  conditions,  will  be  stained  pink,  an  evidence  of  their  beginning 
disintegration.  At  a  later  stage  in  the  process  of  intracellular  digestion, 
the  bacteria  will  lose  their  form,  and  appear  swollen,  granular,  and 
vacuolated,  and  finally  will  be  no  longer  distinguishable.  If,  on  the 
other  hand,  the  ingestion  of  bacteria  brings  about  the  death  of  a  leucocyte 
the  neutral  red  will  not  stain  the  bacteria,  the  digestive  vacuoles  will  not 
form,  and  the  leucocyte  itself  will  disintegrate. 

It  must  not  be  forgotten,  however,  that  not  all  microorganisms  are 
equally  susceptible  to  phagocytosis.  Some  may  resist  ingestion  more 
energetically  than  others  by  agencies  not  fully  understood.  Others 
again,  like  the  tubercle  bacillus  and  the  anthrax  bacillus  for  instance, 
may,  after  ingestion,  oppose  great  difficulties  to  intracellular  digestion. 

To  a  certain  extent,  moreover,  the  variety  of  the  bacterium  deter- 
mines the  variety  of  phagocyte  attracted  to  the  point  of  invasion.  In 
the  cases  of  most  of  the  bacteria  of  acute  diseases,  the  microphages  or 
polymorphonuclear  leucocytes  are  the  ones  upon  which  the  brunt  of 
the  battle  devolves.  Other  invaders,  like  the  Bacillus  tuberculosis, 
blastomyces,  and  others,  find  themselves  opposed  chiefly  by  the  macro- 
phages. Cells  of  animal  origin,  such  as  the  dead  or  injured  cells  of  the 
animals'  own  body  or  the  cells  of  other  animals  artificially  introduced, 
are  ingested  by  macrophages.  This  is  true  also  of  many  parasites  of 
animal  nature. 

It  is  clear,  thus,  that  the  process  of  phagocytosis  is  a  universal  re- 
sponse on  the  part  of  the  body  to  the  invasion  of  foreign  particles  of 
dead  material,  of  alien  cells,  and  of  living  microorganisms.  It  remains 
to  be  shown  upon  what  basis  this  process  may  be  regarded  as  an  essential 
feature  in  protecting  the  body  against  infection. 

The  numerous  researches  of  MetchnikofP  have  brought  out  the 
important  fact  that  phagocytosis  is  regularly  more  active  in  cases  in 


PHAGOCYTOSIS  279 

which  the  infected  animal  or  human  being  eventually  recovers.  In 
animals,  furthermore,  which  show  a  high  natural  resistance  against  any 
given  microorganism,  phagocytosis  is  decidedly  more  energetic  than  it 
is  in  animals  more  susceptible  to  the  same  incitant.  Thus,  experiment- 
ing with  anthrax  infection  in  rats,  Metchnikoff  was  able  to  show  that,  in 
these  animals,  a  decidedly  more  rapid  and  extensive  phagocytosis  of 
anthrax  bacilli  takes  place  than  in  rabbits  and  guinea-pigs  and  other 
animals  which  are  delicately  susceptible  to  this  infection.  While 
different  interpretations  have  been  attached  to  this  phenomenon,  its 
actual  occurrence  may  be  accepted  as  a  proven  fact. 

In  his  later  investigations,  furthermore,  Metchnikoff  was  able  to 
show  that  a  direct  parallelism  existed  between  the  development  of 
immunity  in  an  artificially  immunized  animal  and  the  phagocytic  powers 
of  its  white  cells.  He  showed  that  rabbits  artificially  immunized  to 
anthrax ,  responded  to  anthrax  infection  by  a  far  more  active  phagocy- 
tosis than .  did  noiTaal,  fully  susceptible  animals  of  the  same  species. 

It  is  quite  impossible,  in  the  space  allotted,  to  recount  the  many 
similar  experiments  by  which  the  accuracy  of  these  observations  has  been 
confirmed.  While  few  bacteriologists  at  the  present  day  harbor  any 
doubt  as  to  the  truth  of  these  contentions,  the  fundamental  differences 
between  the  conclusions  drawn  from  these  various  phenomena  by  the 
school  of  Metchnikoff  and  by  that  of  the  German  workers  may  be 
clearly  stated  as  follows:  Metchnikoff  believes  that  phagocytosis  is 
the  cardinal  factor  which  determines  immunity,  while  Pfeiffer  and 
others  maintain  that  the  determining  factors  upon  which  recovery  or 
lethal  outcome  depends,  lie  in  the  fluids  of  the  body,  the  serous  exudate 
and  its  contents  of  immune  body  and  complement,  while  the  phagocy- 
tosis occurring  coincidently,  is  merely  a  means  of  removal  of  the  bacteria 
after  the  outcome  has  already  been  decided. 

In  the  further  developments  of  his  theory,  Metchnikoff  has  claimed 
that  the  immune  body  and  complement — the  presence  of  which  in 
blood  serum  and  exudates  he  by  no  means  overlooks — are  derivatives 
of  the  leucocytes. 

The  immune  body  or  "fixator,"  as  Metchnikoff  has  named  it,  has 
been  shown  by  W^assermann  and  Takaki^  to  be  most  plentiful  in  the 
spleen,  lymph  nodes,  and  bone  marrow  of  animals — all  of  them  organs  in 
which  large  collections  of  leucocytic  elements  are  found.  Metchnikoff's 
opinions  as  to  the  leucocytic  origin  of  the  complement,  or  "cytase," 

» Wassermann  und  Takaki,  Berl.  klin.  Woch.,  1898. 


280  INFECTION  AND  IMMUNITY 

have  found  support  in  the  experiments  of  Levaditi/  who  was  able  to 
demonstrate  the  absence  of  complement  in  blood  plasma, — i.e.^  where  no 
destruction  of  leucocytes  had  taken  place — and  in  those  of  Cantucuz^ne/ 
who  showed  that  cholera-immune  guinea-pigs  would  succumb  to  intra- 
peritoneal injection  of  these  bacteria  when  the  diapedesis  of  leucocytes 
had  been  prevented  by  the  administration  of  opium.     •      ~ 

The  chapter  of  phagocytosis  in  its  relation  to  bacterial  immunity  is 
by  no  means  closed.  The  problems  involved  in  it  are  intricate  and  will 
require  much  further  study.  The  subsequent  sections  upon  opsonins, 
aggressins,  and  upon  leucocyte  extract,  incorporate  the  more  recent 
studies  which  may  be  said  to  have  followed  logically  in  the  footsteps 
of  Metchnikoff's  work. 

» Levaditi,  Presse  med.,  1900. 

«  Cantacuzene,  Ann.  de  Tinst.  Pasteur,  1897 


CHAPTER  XVIII 

OPSONINS,  LEUCOCYTE   EXTRACT,   AND    AGGRESSINS 
OPSONINS 

ALTHough  the  theories  of  immunity  are,  as  we  have  stated,  generally 
classified  as  the  humoral  and  the  cellular  or  phagocytic  theories,  the 
separation  has  never,  even  in  the  minds  of  the  warmest  partisans,  been 
an  absolute  one.  Thus,  Buchner  and  his  successors  looked  for  the 
origin,  first,  of  alexin,  then  of  complement,  in  the  leucocytes,  and 
Metchnikoff  attributed  to  immune  serum  the  quality  of  stimulating  the 
leucocytes  (stimulins)  to  increased  phagocytosis.  The  serum,  accord- 
ing to  Metchnikoff,  acted,  not  directly  upon  the  bacteria,  in  the  nature 
of  bactericidal  or  lytic  substances,  but  rather  upon  the  leucocytes,  pre- 
paring or  arming  these  for  the  fray.  Denys  and  Leclef  ^  were  the  first 
definitely  to  oppose  this  view.  These  authors,  on  the  basis  of  ex. 
periments  done  upon  streptococcus  immunity  in  rabbits,  came  to  thQ 
conclusion  that  the  serum  aided  phagocytosis  rather  by  its  action  upon 
the  bacteria  than  by  its  influence  upon  the  leucocytes. 

Wright  2  in  1903  and  1904  undertook  a  systematic  study  of  the  re- 
lation of  the  blood  serum  to  phagocytosis,  in  a  series  of  careful  experi- 
ments. Using  his  own  modifications  of  the  technique  of  Leishman,^ 
he  first  determined  the  direct  dependence  of  phagocytosis  upon  some 
substance  contained  in  the  blood  serum.  He  further  proved  conclu- 
sively that  this  serum  component  acts  upon  the  bacteria  directly  and 
not  upon  the  leucocytes,  is  bound  by  the  bacteria,  and  renders  them 
subject  to  phagocytosis.  The  presence  of  these  substances  in  sera, 
furthermore,  which  appear  entirely  free  from  bactericidal  or  lytic 
bodies,  and  the  thermolabile  character  of  the  substances  (60°  for  ten 
or  fifteen  minutes  destroys  them)  seemed  to  exclude  their  identity 
with  the  immune  bodies  of  other  authors. 

1  Denys  et  Ledef,  La  cellule,  xi,  1895. 

2  Wright  and  Douglas,  Proc.  Royal  Soc.  London,  Ixxii,  1904, 
■heishman,  Brit.  Med.  Jour.,  i,  1902. 

19  281 


282  INFECTION  AND  IMMUNITY 

Because  of  their  action  in  preparing  the  bacteria  for  ingestion  by  the 
leucocytes,  he  named  these  bodies  '^  opsonins  "  {pilxoviio^  to  prepare  food). 

Neufeld  and  Rimpau^  soon  after,  and  independently  of  Wright, 
described  similar  substances  in  the  blood  serum  of  streptococcus  and 
pneumococcus  immune  animals,  which  they  called  bacteriotropins. 
Because  of  their  greater  thermostability  it  is  not  yet  possible  to  identify 
these  bacteriotropins  absolutely  with  the  opsonins. 

The  importance  of  these  opsonic  substances  in  immunity  was  shown 
by  Wright  ^  in  a  series  of  experiments  in  which  he  determined  that  in 
pv^rsons  ill  with  staphylococcus  or  tubercle-bacillus  infections,  the  phago- 
cytic powers  were  relatively  diminished  toward  these  microorganisms, 
but  could  be  specifically  increased  upon  active  immunization  with  dead 
bacteria  or  bacterial  products. 

The  results  of  Wright  have  been  confirmed  and  elaborated  by  nu- 
merous workers. 

The  diminished  power  of  leucocytes  to  take  up  bacteria  without  the 
co-operation  of  serum  was  demonstrated,  after  Wright,  by  Hektoen  and 
Ruediger,^  who  worked  with  gradually  increasing  dilutions  of  serum. 
The  contention  of  the  Wright  school,  however,  that  leucocytes  are  en- 
tirely impotent  for  phagocytosis  without  the  aid  of  serum,  can  not  be 
regarded  as  proven,  in  face  of  the  work  of  Lohlein  *  and  others  who 
have  observed  phagocytosis  on  the  part  of  washed  leucocytes. 

The  specificity  of  opsonins  and  their  multiplicity  in  a  given  serum 
were  shown  mainly  by  the  work  of  Bullock  and  Atkin,^  Hektoen  and 
Ruediger,^  and  Bullock  and  Western.^  These  authors  showed  that  the 
opsonic  substances  in  sera  could  be  absorbed  out  of  the  sera,  one  by  one, 
by  treatment  with  various  species  of  bacteria,  a  procedure  analogous  to 
the  method  of  absorption  used  in  the  study  of  agglutinins. 

The  increase  of  phagocytic  power  demonstrated  by  Wright  in  immune 
sera  naturally  led  to  the  question  whether  this  depended  merely  upon 
an  increase  of  the  normal  opsonins  or  whether  the  newly  formed  immune 
opsonins  were  entirely  different  substances.  The  greater  thermosta- 
bility of  the  opsonins  in  immune  sera  seemed,  at  first,  to  support  the 

1  Neufeld  und  Rimpau,  Deut.  med.  Woch.,  xl,  1904. 

2  Wright  and  Douglas,  Proc.  Roy.  Soc,  London,  Ixxiv,  1905 

3  Hektoen  and  Rv^diger,  Jour.  Inf.  Dis.,  ii,  1905. 

»  Lohlein,  Ann.  de  Tinst.  Pasteur,  1905  and  1906. 

•  Bullock  and  Atkin,  Proc.  Roy.  Soc,  London,  body,  1905. 

6  Hektoen  and  Ru^diger,  loc.  cit. 

»  Bullock  and  Western^  Proc.  Roy.  Soc.,  loc.  dt. 


OPSONINS  283 

latter  view.  Dean,*  however,  showed  that  not  all  of  the  normal  opsonins 
are  thermolabile  and  that,  by  absorption  experiments,  bacteria  treated 
with  normal  seia  could  be  prevented  from  taking  up  opsonins  from 
immune  sera.  These  facts  seem  to  point  strongly  toward  the  identity  of 
normal  and  immune  opsonic  substances. 

Further  study  of  the  opsonins  has  led  to  numerous  other  questions 
regarding  their  structure,  their  relation  to  other  immune  bodies,  etc.. 
which  are  largely  still  in  the  stage  of  controversy,  and  for  which  the 
original  monographs  must  be  consulted. 

The  controversial  questions  may  be  briefly  reviewed  as  follows: 

As  stated  above,  Wright  believed  originally  that  the  bodies  dis- 
covered by  him  in  normal  sera,  the  "normal  opsonins/^  in  other  words, 
were  distinct  bodies  that  could  not  be  identified  with  either  the  comple- 
ment or  antibodies  present  in  serum.  Neufeld  and  Hiine,^  Levaditi  and 
Inmann,^  and  others,  on  the  other  hand,  maintain  that  the  opsonic 
action  of  normal  serum,  at  least,  is  intimately  related  to  the  complement 
contents  of  such  serum. 

They  base  this  contention  not  only  upon  the  thermolability  of  normal 
opsonins,  but  also  upon  the  fact  that  opsonin  may  be  removed  from 
normal  serum  at  the  same  time  as  complement  by  the  method  of  com- 
plement fixation,  detailed  in  another  section  (see  pp.  245  and  261).^ 

The  contention  of  Wright  that  the  thermostable  opsonic  substances 
of  immune  serum  are  distinct  bodies,  not  identical  with  the  ambocep- 
tors, is  supported  by  the  work  of  Hektoen,^  Neufeld  and  Topfer,® 
and  others.  The  problem,  however,  can  by  no  means  be  regarded  as 
finally  settled,  since  other  workers,  notably  Levaditi,  are  inclined  to 
identify  the  immune  opsonins  with  lytic  amboceptors. 

As  to  the  structure  of  the  opsonic  substances,  moreover,  dif- 
ferences of  opinion  still  exist.  Hektoen  and  Ruediger  '  who  have 
investigated  the  question  attribute  to  opsonins  a  complex  constitution. 
They  believe  them  to  possess  a  thermostable  haptouhore  group 
and  a  thermolabile  "  opsonophore ''  group  and  that  heating  beyond 
a   definite    temperature    converts    the    opsonins    into    opsonoids    by 

1  Dean,  Proc.  Roy.  Soc,  London,  Ixxvi,  1905. 

2  Neufeld  and  Hiine,  Arb.  a.  d.  kais.  Gesundheitsamt,  xxv. 

»  Levaditi  and  Inmann,  Compt.  rend,  de  la  soc.  de  biol.,  62,  1907. 

*  Levaditi,  Presse  m^dicale,  70,  1907. 

8  Hektoen,  Jour,  of  Inf.  Dis.,  iii,  1906. 

«  Neufeld  und  ToTpfer,  Cent.  f.  Bakt.,  xxxviii,  1905. 

» Hektoen  and  Rwdigerf  Jour,  of  Inf.  Dis.,  ii  1905. 


284  *         INFECTION  AND  IMMUNITY 

destruction  or  alteration  of  the  ''opsonophore"  group.  This  view  is 
not  shared  by  all  workers  and  has  been  disputed  by  Bullock  and  Atkin.^ 
The  Technique  of  Wright. — The  three  factors  necessary  for  the  per- 
formance of  an  opsonic  test  are  (1)  the  blood  serum  to  be  tested;  (2) 
an  even  emulsion  of  bacteria,  and  (3)  leucocytes. 

(1)  Blood  serum  is  obtained  by  bleeding  from  the  finger  and  receiving 
the  blood  into  glass  capsules  (Fig.  66).  These  are  sealed  at  both  ends; 
the  blood  is  allowed  to  clot;  and  the  separation  of  serum  is  hastened  by 
a  few  revolutions  of  a  centrifuge. 

(2)  The  bacterial  emulsion  is  obtained  by  rubbing  up  a  few 
loopfuls  of  a  twenty-four-hour  slant  agar  culture  with  a  little  phj^sio- 
logical  salt  solution  (0.85  per  cent)  in  a  watch  glass.     A  very  small 

amount  of  salt  solution  is  used  at  first 
and  more  is  gradually  added,  drop  by 
drop,  as  the  emulsion  becomes  more 
even.  The  final  breaking  up  of  the 
smaller  clumps  is  best  accomplished  by 

„  ^.         ,     ^  cutting  off  very  squarely  the  end  of  a 

Fig.   66.— Wright's    Capsule    for  .„  ^  ...■,. 

Collecting  Blood.  capillary    pipette,   placing   it   perpen- 

dicularly against  the  bottom  of  the 
watch  glass,  and  sucking  the  emulsion  in  and  out  through  the  narrow 
chink  thus  formed.     (Fig.  67.) 

Emulsions  of  tubercle  bacilli  are  more  difficult  to  make.  The  bacilli 
filtered  off  in  the  manufacture  of  old  tuberculin  are  commonly  used. 
These  are  washed  in  salt  solution  on  the  filter,  and  are  then  scraped  off 
and  sterilized.  They  are  then,  in  a  moist  condition,  placed  in  a  mortar 
and  thoroughly  ground  into  a  paste.  While  grinding,  salt  solution 
1.5  cent)  is  gradually  added  until  a  thick  emulsion  appears.  This 
emulsion  may  be  diluted  and  larger  clumps  '  separated  by  centri  - 
fugalization. 

(3)  The  leucocytes  are  obtained  by  bleeding  from  the  ear  or  finger 
directly  into  a  solution  containing  eighty-five  hundredths  per  cent  to  one 
per  cent  of  sodium  chlorid  and  five-tenths  to  one  and  five-tenths  per  cent 
of  sodium  citrate.  Ten  or  fifteen  drops  of  blood  to  5  or  6  c.c.  of  the 
solution  will  furnish  sufficient  leucocytes  for  a  dozen  tests.  This 
mixture  is  then  centrifugalized  at  moderate  speed  for  five  to  six  minutes. 
At  the  end  of  this  time,  the  corpuscles  at  the  bottom  of  the  tube 
will  be  covered  by  a  thin  grayi^  pellicle,  the  buffy  coat,  consisting 

1  Bullock  and  Atkin,  Proc.  Royal  Soc,  Ixxiv,  1905. 


OPSONINS  285 

chiefly  of  leucocytes.  These  are  pipetted  off  with  a  capillary  pipette 
(by  careful  superficial  scratching  movements  over  the  surface  of  the 
buffy  coat). 

There  being,  of  course,  no  absolute  scale  for  phagocytosis,  whenever 
an  opsonin  determination  is  made  upon  an  unknown  serum,  a  parallel 
control  test  must  be  made  upon  a  normal  serum.  This  normal  is  best 
obtained  by  a  "  pool  '^  or  mixture  of  the  sera  of  five  or  six  supposedly 
normal  individuals. 

The  three  ingredients — serum,  bacterial  emulsion,  and  leucocytes — 
having  thus  been  prepared,  the  actual  test  is  carried  out  as  follows: 


Fig.  67. — Pipette  for  Opsonic  Work. 

Capillary  pipettes  of  about  six  or  seven  inches  in  length  and  of  nearly 
even  diameter  throughout,  are  made.  These  are  fitted  with  a  nipple 
and  a  mark  is  made  upon  them  with  a  grease-pencil  about  2  to  3  cm. 
from  the  end  (Fig.  68).  Corpuscles,  bacteria,  and  serum  are  then 
successively,  in  the  order  named,  sucked  into  the  pipette  up  to 
the  mark,  being  separated  from  each  other  by  small  air-bubbles.  Equal 
quantities  of  each  having  thus  been  secured,  they  are  mixed  thoroughly 
by  repeatedly  drawing  them  in  and  out  of  the  pipette  upon  a  slide. 
The  mixture  is  then  drawn  into  the  pipette;  the  end  is  sealed;  and 
incubation  at  37.5°  is  carried  on  for  an  arbitrary  time,  usually  fifteen 
to  thirty  minutes.^    The  control  with  normal  serum  is  treated  in  exactly 


Fig.  68. — Pipette  with  three  Substances,  Corpuscles,  Bacteria,  and  Serum, 

AS  first  taken  up. 

ttie  same  way.  After  incubation  the  end  of  the  pipette  is  broken  off, 
the  contents  are  again  mixed,  and  smears  are  made  upon  glass  slides  in 
the  ordinary  manner  of  blood  smearing.  Staining  may  be  done  by 
Wright's  modification  of  Leishman's  stain,  by  Jenner's,  or  by  any  other 
of  the  usual  blood  stains.  In  these  smears,  then,  the  number  of  bacteria 
contained  in  each  leucocyte  is  counted.     The  contents  of  about  eighty 

^  For  the  purpose  of  incubation,  specially  constructed  water  baths,  marketed  under 
the  name  of  "  opsonizers,"  may  be  used. 


286  INFECTION  AND  IMMUNITY 

to  one  hundred  cells  are  usually  counted  and  an  average  is  taken. 
This  average  number  of  bacteria  in  such  leucocytes  is  spoken  of  a^  the 
"phagocytic  index."  The  phagocytic  index  of  the  tested  serum,  divided 
by  that  of  the  "normal  pool"  (control)  serum,  gives  the  ''opsonic 
index." 

Another  method  of  estimating  the  opsonic  content  of  a  given  blood 
serum  has  been  contributed  by  Simon,  Lamar,  and  Bispham.^  These 
authors  employed  dilutions  both  of  the  patient's  serum  and  of  normal 
serum  ranging  from  one  in  ten  to  one  in  one  hundred.  With  these 
dilutions,  they  carry  out  opsonic  experiments  "with  bacterial  emulsions 
and  washed  leucocytes  in  the  same  way  as  this  is  done  in  the  Wright 
method,  except  that  they  recommend  the  employment  of  thinner  bac- 
terial emulsions  than  are  usually  employed  in  the  former  method.  In 
examining  their  slides,  they  do  not  estimate  the  number  of  bacteria 
found  within  the  leucocytes,  but  rather  the  percentage  of  leucocytes 
which  actually  take  part  in  the  phagocytic  process,^  i.e.,  which  con- 
tain bacteria. 

By  the  same  method  of  dilution,  they  determine  what  they  have 
called  ''the  opsonic  coefficient  of  extinction,"  a  phrase  which  is  used  to 
express  the  degree  of  dilution  of  the  serum  at  which  no  further  phagocy- 
tosis takes  place.  They  claim  for  their  methods  the  more  dehcate  de- 
termination of  variations  in  opsonic  power.  The  method  has  not  been 
sufficiently  used  to  permit  the  expression  of  an  opinion  as  to  its  value. 

The  Vaccine  Therapy  of  Wright. — In  connection  with  his  more 
theoretical  work  upon  opsonins,  Wright  has  laid  much  stress  upon  the 
value  of  active  immunization  in  the  treatment  of.  infectious  diseases. 
Beginning  his  work  with  staphylococcus  and  tubercle-bacillus  infections, 
he  has  extended  his  methods,  with  the  aid  of  many  collaborators,  to 
gonococcus,  streptococcus,  pneumococcus,  and  a  number  of  other 
bacterial  infections.  In  all  these  cases,  when  possible,  he  uses  for 
therapeutic  purposes  a  so-called  "autogeiious  vaccine"  which  is  made 
with  the  bacteria  isolated  from  the  patient  himself.  In  the  case  of 
tubercle-bacillus  infections,  he  uses  for  treatment  the  new-tuberculin- 
bacillar3^-emulsion  of  Koch.  The  production  of  vaccine  is,  according 
to  Wright,  as  follows: 

Production  of  Vaccines. — After  isolation  of  the  organisms  from  the 
patient,  cultures  are  made  with  a  view  of  obtaining  considerable  amounts 

^  Simon,  Lamar,  and  Bispham,  Jour.  Exp.  Med.,  viii,  1906. 
9 Simon  and  Lamar,  Johns  Hopkins  Hosp.  Bull.,  xvii,  1906. 


OPSONINS  287 

of  bacterial  growth.  In  making  vaccines  with  poorly  growing  organisms, 
large  surfaces  must  be  inoculated.  Organisms  are  best  grown  for  this 
purpose  upon  the  surface  of  agar  or  glucose  agar  (the  enrichment  of  the 
agar  with  sugar  or  acetic  fluid,  etc.,  depending  upon  the  cultural  require- 
ments of  the  organism  in  question),  in  square  eight-ounce  medicine 
bottles  laid  upon  their  sides.  This  furnishes  a  large  area  for  inoculation. 
After  sufficient  growth  has  taken  place  upon  the  agar,  two  or  three  cubic 
centimeters  of  sterile  normal  salt  solution  are  introduced  into  the 
bottles  with  a  sterile  pipette.  With  this  the  growth  is  gently  washed 
off  the  surface  of  the  agar,  more  salt  solution  gradually  being  added  as 
necessary.  The  emulsification  may  be  facilitated  by  gently  scraping 
the  growth  off  the  medium  by  means  of  a  flexible  platinum  loop.  This 
thick  bacterial  emulsion  is  then  pipetted  out  of  the  bottles,  during  which 
process  an  equalization  of  the  emulsion  can  be  attained  by  repeated 
sucking  in  and  out  with  the  pipette.  The  emulsion  is  then  placed  in  a 
sterile  test  tube  which  may  then  be  drawn  oiit  at  its  open  end  into  a 
capillary  opening.  It  is  a  point  of  practical  importance  that,  in  pre- 
paring such  capsules  out  of  a  test  tube,  a  few  inches  of  air  space  should 
be  left  above  the  surface  of  the  emulsion,  so  that  expansion  during 
heating  may  not  blow  out  the  top  of  the  glass  tube.  A  dozen  or  so  of 
sterile  glass  beads  may  be  put  into  these  tubes  in  order  to  aid  in  emul- 
sification. Shaking  the  beads  in  such  a  tube  will  help  in  breaking  up 
email  clumps  of  bacteria. 

The  emulsion  is  then  standardized;  that  is,  a  numerical  estimation 
of  bacteria  per  cubic  centimeter  must  be  made.  This  standardization 
is  best  done  before  sterilization,  because  during  the  latter  process  a 
number  of  bacteria  may  be  broken  up,  and,  while  unrecognizable 
morphologically,  are,  nevertheless,  represented  in  the  emulsion  by  their 
products.  The  standardization  may  be  accomplished  by  highly  diluting 
a  definite  volume  of  the  emulsion,  planting  plates  with  definite  quan- 
tities of  the  dilution,  and  counting  colonies.  Wright  prefers,  as  more 
exact,  an  enumeration  of  the  bacteria  against  red  blood  cells.  This  is 
done  in  the  following  way: 

A  little  of  the  emulsion  is  placed  in  a  w^atch  glass  and  from  it,  with  a 
pipette  1  as  used  in  the  estimation  of  the  opsonic  index,  one  volume  is 
taken  and  is  mixed  with  an  equal  volume  of  blood  from  the  finger  and 
two  or  three  volumes  of  salt  solution.  The  salt  solution  is  added  in 
order  to  dilute  the  red  cells  so  that  they  can  be  conveniently  counted 
and  to  prevent  clotting.  These  substances  are  thoroughly  mixed  in  a 
pipette  and  spread  upon  a  slide  as  in  making  a  blood  smear,  and  as  even 


288  INFECTION   AND  IMMUNITY 

and  uniform  a  smear  as  possible  should  be  made.  They  are  then  stained 
either  by  Jenner^s  or  Wright's  blood  stain. 

The  preparations  are  examined  with  an  oil-immersion  lens.  In 
order  to  limit  a  definite  microscopic  field,  it  is  convenient  to  use  an 
Ehrlich  diaphragm,  or  else,  in  lieu  of  this,  to  mark  a  circle  with  a  blue 
pencil  upon  the  lens  of  the  eye-piece.  The  red  blood  cells  and  bacteria, 
in  a  number  of  these  fields,  are  counted  and  the  ratio  between  them  is 
estimated.  Knowing  the  number  of  red  blood  cells  to  the  cubic  milli' 
meter  in  the  particular  blood  employed,  by  previous  blood  counts 
and  knowing  that  equal  volumes  of  blood  and  of  bacterial  emulsion 
have  been  used  in  the  mixture,  it  is  easy  from  this  ratio  to  ascertain 
the  number  of  bacteria  contained  in  a  cubic  millimeter  of  the  original 
emulsion.  Thus,  for  instance,  if  in  an  average  of  twenty  fields  bacteria 
are  to  red  blood  cells  as  two  is  to  one,  and  the  blood  employed  con- 
tains five  million  red  blood  cells  to  each  cubic  millimeter,  then  a  cubic 
millimeter  of  our  emulsion  contained  ten  million  bacteria,  and  a  cubic 
centimeter  one  thousand  times  as  many. 

Special  centrifuge  tubes  with  graduated  narrow  tips  at  the  bottom 
have  been  suggested  by  Hopkins  for  vaccine  standardization.  Bacteria 
centrifugalized  up  to  a  certain  mark  represent  a  definite  number  per 
cu.  mm.  when  taken  up  in  a  given  volume  of  salt  solution. 

The  vaccine,  thus  standardized,  is  sterilized  at  60°  C.  for  one  hour 
for  several  days.  Its  sterility  is  then  controlled  by  culture  and  animal 
inoculation. 

From  this  stock  emulsion  small  quantities  may  be  drawn  off  and 
diluted  for  therapeutic  use. 

The  initial  dose  given  by  Wright  in  staphylococcus  infections,  in 
which  the  method  has  been  most  frequently  employed,  varies  from 
fifty  to  one  hundred  millions  of  bacteria.  In  working  with  the  tubercle 
bacillus,  the  ordinary  tuberculin  dosage  is  adhered  to. 

Wright,  in  his  work,  makes  use  of  the  opsonic  index  in  order  to 
estimate  changes  in  the  resistance  of  the  patient  against  the  given  in- 
fection. In  other  words,  he  bases  his  judgment  as  to  whether  the 
patient  is  improving  or  not,  upon  the  opsonic  power  of  the  patient's 
serum.  In  following  the  opsonic  index  of  a  patient  during  systematic 
treatment  with  vaccine,  Wright  has  found  definite  changes  upon  the 
basis  of  which  he  constructs  a  curve  of  opsonic  power.  Immediately 
after  the  injection  of  vaccine,  he  finds  that  there  is  a  brief  period  during 
which  the  opsonic  power  of  the  patient  is  depressed  below  its  original 
state.  This  he  calls  the  negative  phase.  The  length  of  time  occupied 
by  this  negative  phase  depends  both  upon  the  condition  of  the  patient 


1 


LEUCOCYTE  EXTRACT  289 

iand  upon  the  size  of  the  dose  given.  It  is  usually  completed  within 
twenty-four  hours.  After  this,  there  is  a  gradual  rise  in  the  opsonic 
power,  at  first  rapid,  later  more  slow,  until  a  maximum  is  reached  after  a 
varying  number  of  days.  This  period  of  rise  represents  the  positive 
phase.  The  second  inoculation  with  vaccine  should,  according  to  Wright, 
be  made  when  the  opsonic  power  is  again  beginning  to  sink  after  the 
highest  point  of  the  positive  phase. 

The  facts  of  Wright's  investigations  have  been  given  in  the  pre- 
ceding without  much  critical  consideration.  Those  features  which  con- 
cern themselves  with  the  proof  of  the  opsonic  properties  of  normal 
and  immune  serum  have  been  of  the  greatest  scientific  importance  and 
a  great  deal  of  benefit  has  accrued  from  the  renewed  attention  turned 
by  him  toward  methods  of  active  immunization  in  human  beings. 
Vaccine  therapy  in  many  conditions  has  come  to  stay,  although  some 
of  the  very  extravagant  earlier  claims  have  had  to  fall  down.  It 
is  also  pretty  certain  at  present  that  the  opsonic  index  measure- 
ments as  a  guide  to  treatment  is  of  very  little  value  and  may  even  mis- 
lead. 

Leucocyte  Extract. — In  the  sections  upon  Phagocytosis  and  Op- 
sonins, we  have  discussed  the  protective  action  exerted  by  the  living 
leucocytes  against  bacterial  infection  and  the  relation  of  these  cells  to 
the  blood  serima;  furthermore,  that,  while  our  knowledge  of  the  blood 
serum,  as  developed  at  present,  shows  that  phagocytes  may  be  aided 
by  this  in  the  ingestion  of  bacteria,  the  subsequent  digestion  of  the 
germs,  and  possibly  the  neutralization  or  destruction  of  their  intracel- 
lular poisons,  is,  as  far  as  we  know,  largely  accomplished  by  the  unaided 
phagocytic  cell.  It  is  an  obvious  thought,  therefore,  that,  in  the  struggle 
with  bacterial  invaders,  the  leucocytic  defenders  might  be  considerably 
re-enforced  if  they  were  furnished,  as  directly  as  possible,  with  a  further 
supply  of  the  very  weapons  which  they  were  using  in  the  fight  with 
the  microorganisms.  With  this  thought  as  a  point  of  departure.  Hiss  ^ 
conceived  the  plan  of  injecting  into  infected  subjects  the  substances 
composing  the  chief  cells  or  all  the  cells  usually  found  in  exudates,  in 
the  most  diffusible  form  and  as  Httle  changed  by  manipulation  as  possi- 
ble ;  and  he  also  assumed  that  extracts  would  be  more  efficacious  than 
living  leucocytes  themselves,  since  if  diffusible  they  would  be  distrib- 
uted impartially  to  all  parts  of  the  body  by  the  circulatory  mechan- 
ism. They  would  then,  as  quickly  as  absorption  would  permit,  relieve 
the  fatigued  leucocyte  and  also  protect  by  any  toxin-neutralizing  oi 
other  power  they  might  possess,  the  cells  of  highly  specialized  functions. 

...    — — .1  .» 

» Hiss,  Jour,  M^a.  Res..  N.  S.,  xiv,  3. 1908. 


290  '  INFECTION  AND  IMMUNITY 

The  method  of  obtaining  these  substances  as  used  both  in  animal 
experiments  and  in  the  treatment  of  human  subjects  is  at  present  as 
follows: 

Rabbits,  preferably  of  1,500  grams  weight  or  heavier,  receive  intra- 
pleural injections  of  aleuronat.  This  is  prepared  by  making  a  three 
per  cent  solution  of  starch  in  meat-extract  broth,  without  heating, 
and  adding  to  this,  after  the  starch  has  gone  into  thorough  emulsion, 
five  per  cent  of  powdered  aleuronat.  This  is  thoroughly  mixed,  boiled 
for  five  minutes,  and  filled  into  sterile  potato  tubes,  20  c.c.  into  each  tube. 
Final  sterilization  is  done  preferably  in  an  autoclave.  The  rabbit  in- 
jections are  carried  out  by  injecting  10  c.c.  into  each  pleural  cavity 
in  the  intercostal  spaces  at  the  level  of  the  end  of  the  sternum,  in  the 
anterior  axillary  line,  great  care  being  exerted  to  avoid  puncturing  of 
the  lungs.  The  rabbits  are  left  for  twenty-four  hours,  at  the  end  of 
which  time  a  copious  and  very  cellular  exudate  will  have  accumulated 
in  the  pleural  cavities.  This  is  removed,  after  killing  the  animals  with 
chloroform,  by  opening  the  anterior  chest  wall  under  rigid  precautions 
of  sterility,  and  pipetting  the  exudate  into  sterile  centrifuge  tubes. 
Immediate  centrifugalization  before  clotting  can  take  place  then  per- 
mits the  decanting  of  the  supernatant  exudate  fluid.  To  the  leuco- 
cytic  sediment  is  then  added  about  2  c.c.  of  sterile  distilled  water,  and 
the  emulsion  is  thoroughly  beaten  up  with  a  stiff  bent  platinum  spatula. 
Smears  are  now  made  on  slides,  stained  by  Jenner's  blood  stain,  and  ex- 
amined for  possible  bacterial  contamination.  It  is  well  also  to  take 
cultures.  Sterile  distilled  water  is  then  added  to  each  tube,  about  twenty 
volumes  to  one  volume  of  sediment,  and  the  tubes  are  set  away  in  the 
incubator  for  eight  hours.  At  the  end  of  this  time  the  sterility  is  again 
controlled  as  above,  and  further  extraction  in  the  refrigerator  continued 
until  the  extract  is  used. 

In  experimenting  upon  animals.  Hiss  ^  observed  that  pneumococcus, 
staphylococcus,  streptococcus,  meningococcus,  and  typhoid,  dysentery, 
and  cholera  infections  in  rabbits  and  guinea-pigs  were  profoundly  modi- 
fied when  injections  of  leucocyte  extracts,  prepared  as  above,  were  ad- 
ministered intraperitoneally  or  subcutaneously  during  the  course  of  the 
infection.  In  many  cases  animals  were  saved  by  these  substances  from 
infections  which  proved  rapidly  fatal  in  untreated  control  animals,  even 
when  the  protective  injections  were  made  as  late  as  twenty-four  hours 
after  intravenous  infection. 
- 

^Hi$s,  Jour.  Med.  Res.,  N.  S.,  xiv,  3,  1908, 


AGGRESSINS  291 

In  applying  this  method  of  treatment,  by  subcutaneous  injections, 
to  infections  in  man.  Hiss  and  Zinsser  observed  distinctly  beneficial 
results  in  cases  of  epidemic  cerebrospinal  meningitis,  in  lobar  pneu- 
monia, in  staphylococcus  infections,  and  in  erysipelas.^ 

In  experimenting  with  the  leucocyte  extracts  in  vitro  the  same  au- 
thors were  able  to  show  that  precipitates  occurred  when  clear  leucocyte 
extract  and  the  clear  extract  of  various  bacteria  were  mixed.^ 

Further  experiments,  carried  out  both  in  animals  and  in  the  test 
tube,  showed  that  while  the  leucocytic  extracts  possessed  slight  bactericidal 
powers  for  a  variety  of  microorganisms,  these  attributes  did  not  seem 
sufficient  to  explain  the  profound,  modifying  influences  exerted  upon 
bacterial  infections  by  these  extracts.  Hiss  and  the  writer  in  their 
earlier  work  thought  that  possibly  the  action  of  the  leucocytic  extracts 
consisted  of  the  neutralization  of  bacterial  poisons.  It  is  the  opinion 
of  the  writer  at  present  that  it  is  more  likely  that  these  substances 
favorably  influence  infection  by  their  action  in  increasing  leucocytosis, 
or,  in  other  words,  non-specific  chemotactic  action. 

That  bactericidal  substances  can  be  extracted  from  leucocytes  by 
various  methods  has  been  repeatedly  shown  by  Schattenfroh,  Petterson, 
Korschun,  and  others.^  The  researches  of  Petterson  as  well  as,  more 
recently,  the  work  of  Zinsser,  have  shown  that  these  "  endolysins,"  as 
Petterson  has  called  them,  have  a  structure  quite  different  from  that 
of  the  serum  bacteriolysins  in  that  they  are  not  rendered  inactive  by 
temperatures  under  80°  C,  but,  when  once  destroyed  by  higher  tem- 
peratures, can  not  be  reactivated  either  by  the  addition  of  fresh  serum 
or  of  unheated  leucocyte  extracts.  The  last-named  authors,  moreover, 
have  shown  that  these  endocellular  bactericidal  substances  are  not 
increased  by  immunization,  the  quantity  present  in  each  leucocyte  being 
probably  at  all  times  simply  sufficient  for  the  digestion  of  the  limited 
number  of  bacteria  which  can  be  taken  up  by  the  individual  leucocyte. 

The  Problem  of  Virulence. — An  extremely  obscure  chapter  in  our 
knowledge  of  the  reaction  of  animals  and  man  against  infection  is  the 
one  dealing  with  the  questions  of  varying  pathogenicity  between  differ- 
ent bacterial  species  and  between  different  races  of  the  same  micro- 
organism.    We  know  that  certain  bacteria  may  be  injected  into  an 

1  Hiss  and  Zinsser,  Jour.  Med.  Res.,  N.  S.,  xiv,  3,  1908;  ibid.,  xv,  3,  1909. 

2  Hiss  and  Zinsser,  ibid.,  xiv,  3,  1908. 

^Schattenfroh,  Arch.  f.  Hyg.,  1897;  Petterson,  Cent.  f.  Bakt.,  I,  xxxix,  1905,  and 
ibid.,  xlvi,  1908;  Korschun,  Ann.  de  1'  Inst.  Pasteur,  xxii,  1908;  Zinsser,  Jour.  Med. 
Res.,  xxii,  3,  1910. 


292  INFECTION  AND  IMMUNITY 

animal  or  human  being  in  considerable  quantities,  without  producing 
anything  more  than  the  temporary  local  disturbance  following  the  sub- 
cutaneous administration  of  any  innocuous  material.  Other  bacteria, 
on  the  other  hand,  such  as  the  bacillus  of  anthrax  or  the  bacillus  of 
chicken  cholera,  injected  in  the  most  minute  dosage,  may  give  rise  to 
a  rapidly  fatal  septicemia.  Within  the  same  species,  furthermore, 
fluctuations  in  virulence  may  take  place  which  may  depend  upon  a 
variety  of  influences  which  have  been  discussed  in  another  section  and 
need  not  be  recapitulated.  Suffice  it  to  say  that  variations  in  the  sus- 
ceptibility of  inoculated  subjects  do  not,  in  any  way,  furnish  a  sufficient 
explanation  for  these  phenomena. 

In  an  effort  to  cast  light  upon  this  subject.  Bail,  following  in  the 
footsteps  of  his  predecessors,  Kruse,^  Deutsch  and  Feistmantel,^  has 
formulated  his  so-called  "aggressin-theory.'^ 

Bail  ^  was  first  led  to  the  formulation  of  his  theory  by  extensive  re- 
searches which  he  had  made  in  conjunction  with  Petterson  ^  into  an- 
thrax immunity.  He  had  noted,  as  others  before  him  had,  that  animals, 
highly  susceptible  to  anthrax,  often  possessed  marked  bactericidal 
powers  against  this  bacillus.  When  such  animals,  whose  serum  should 
surely  be  capable  of  bringing  about  the  death  of,  at  least,  a  few  hundred 
anthrax  bacilli,  were  injected  with  doses  far  less  than  this  number  they 
nevertheless  succumbed  rapidly  and  the  bacilli  multiplied  enormously 
in  their  bodies.  He  argued  from  this  that  the  injected  microorganisms 
must  possess  some  weapon  whereby  they  were  enabled  to  counteract 
the  protective  forces  of  the  animal  organism.  In  an  anthrax-immune 
animal,  as  a  matter  of  fact,  no  proliferation  of  bacteria  took  place  and 
the  injected  germs  were  rapidly  disposed  of  by  the  protective  forces, 
foremost  of  which  was  phagocytosis. 

The  theory  of  Bail  ^  contains  the  following  basic  principles:  ^ 

Pathogenic  bacteria  differ  fundamentally  from  non-pathogenic  bac- 
teria in  their  power  to  overcome  the  protective  mechanism  of  the  ani- 
mal body,  and  to  proliferate  within  it.  They  accomplish  this  by  virtue 
of  definite  substances  given  off  by  them,  probably  in  the  nature  of  a 
secretion,  which  acts  primarily  by  protecting  them  against  phagocy- 

^  Kruse,  Ziegler's  Beitrage,  xii,  1893. 

2  Deutsch  und  Feistmantel,  "Die  Impfstoffe  in  Sera,"  Leipzig,  1903. 

^Bail,  Cent.  f.  Bakt.,  I,  xxvii,  1900,  and  xxxiii,  1902. 

^Bail  und  Petterson,  Cent.  f.  Bakt.,  I,  xxxiv,  1903;  xxxv,  1904;  xxxvi,  1904. 

^Bail,  Arch.  f.  Hyg.,  lii,  1905;  liii,  1905;  Wien.  klin.  Woch.,  xvii,  1905. 

^Bail  und  Weil,  Wien.  klin.  Woch.,  ix,  1906;  Cent.  f.  Bakt.,  I,  xl,  1906;  xlii,  1906. 


PROBLEM  OF  VIRULENCE  293 

tosis.  These  substances  (referred  to  by  Kruse  as  "Lysins")  were  named 
by  Bail,  "Aggressins.'*  The  production  of  aggressins  by  pathogenic 
germs  is  probably  absent  in  test-tube  cultures,  or,  at  any  rate,  is  greatly 
depressed  under  such  conditions,  but  is  called  forth  in  the  animal  body 
by  the  influences  encountered  after  inoculation. 

These  aggressins  can  be  found,  according  to  Bail,  in  the  exudates 
about  the  site  of  inoculation  in  fatal  infections.  He  obtained  them, 
separate  from  the  bacteria,  by  the  centrifugation  and  subsequent  de- 
canting of  edema  fluid,  and  pleural  and  peritoneal  exudates. 

Two  experimental  observations  are  brought  by  Bail  in  support  of 
the  truth  of  his  contentions.  In  the  first  place,  he  was  able  to  show 
that  fatal  infection  could  be  produced  in  animals  by  the  injection  of 
sublethal  doses  of  bacteria,  when  these  were  administered  with  a  small 
quantity  of  ''aggressin."  He  inferred  from  this  that  the  injected  ag- 
gressin  had  paralyzed  the  onslaught  of  phagocytic  and  other  protective 
agencies,  and  had  thus  made  it  possible  for  the  bacteria  to  proliferate. 

The  second  experimental  support  of  Bail's  theory  consists  in  the 
successful  immunization  of  animals  with  aggressin.  Animals  were 
treated  with  aggressive  exudates,  from  which  all  bacteria  had  been  re- 
moved by  centrifugalization  and  which  had  been  rendered  sterile  by 
three  hours'  heating  to  60°  C.  and  addition  of  0.5%  phenol.  Animals 
so  treated  were  not  only  immune  themselves,  but  contained  a  substance 
in  their  serum  which  permitted  the  passive  immunization  of  other  un- 
treated animals.  Bail  explained  this  by  assuming  the  production  of 
anti-aggressins  in  the  treated  subjects.  His  experiments  and  those  of 
his  pupils  were  conducted  with  the  typhoid  and  dysentery  bacilli,  the 
bacilli  of  chicken  cholera  and  of  plague,  the  cholera  spirillum,  and  va- 
rious micrococci.  According  to  whether  a  microorganism  is  capable  of 
producing  an  aggressin  and  consequently  of  invading  the  animal  body, 
he  divides  bacteria  into  ^'pure  pa,rasites,"  "half  parasites,"  and  "sapro- 
phytes." 

The  theory  of  Bail  has  been  attacked  chiefly  by  Wassermann  and 
Citron,^  Wolff, ^  and  Sauerbeck.^  The  criticism  which  these  investigators 
make  of  Bail's  views  has  succeeded  in  placing  the  "aggressin"  theory 
in  doubt.  It  is  claimed  by  them  that  much  of  the  "aggressive"  char- 
acter of  Bail's  exudates  is  due  to  their  containing  liberated  bacterial 
poisons  (endotoxins).     This  they  have  maintained  both  because  the 


^  Wassermann  and  Citron,  Deut.  med.  Woch.,  xxviii,  1905. 

2  Wolff,  Cent,  f .  Bakt.,  I,  xxxviu,  1906.  «  Sauerbeck,  Zeit.  f .  Hyg.,  Ivi,  1907. 


294  INFECTION  AND  IMMUNITY 

sierile  "aggressin"  exudates  could  be  shown  to  possess  a  considerable 
degree  of  toxicity  and  because  the  aggressive  action  could  be  duplicated 
by  aqueous  extracts  of  bacteria.  Citron/  was  able  to  show,  by  the 
Bordet-Gengou  method  of  complement  fixation,  that  the  exudates  of 
Bail  contained  quantities  of  free  bacterial  receptors,  which,  in  taking 
up  immune  body,  would  neutralize  any  destructive  power  on  the  part 
of  the  infected  animal. 

The  writer  in  conjunction  with  Dwyer^  has  done  certain  experiments 
which  seem  to  indicate  that  Bail's  aggressin  may  be  in  the  nature  of 
anaphylatoxin.  The  addition  of  such  anaphylatoxin  to  bacteria  will 
convert  a  sublethal  into  a  lethal  dose,  as  will  Bail's  aggressin,  and  in 
principle  the  manner  of  production  is  the  same.  The  nature  of  the 
immunity  produced  in  animals  by  Bail's  method  of  treatment  is  less 
easily  explained  and  less  exposed  to  adverse  criticism.  Whatever  may 
be  the  truth  about  the  possession  of  offensive  weapons  on  the  part  of 
bacteria,  it  is  certainly  a  fact  that  microorganisms  differ  much  in  their 
powers  of  defense  against  destruction  by  the  cells  in  sera  of  the  animal 
body.  Virulent  bacteria  are  not  destroyed  by  serum  or  agglutinated 
or  taken  up  by  leucocytes  as  easily  as  are  the  non-virulent.  In  some 
cases  there  seems  to  be  no  morphological  clue  to  the  reason  for  this. 
In  other  cases,  like  pneumococci,  Friedlander  bacilli  and  others,  there 
is  a  bacterial  capsule  which  seems  to  insulate  these  organisms  against 
attack.  Many  bacteria  lose  their  capsules  in  the  non-virulent  stage  on 
culture  media,  but  form  them  within  the  animal  body  in  the  process 
of  infection.  Again,  bacteria  rendered  non-virulent  by  cultivation  on 
artificial  media  may  become  virulent,  inagglutinable,  and  more  resistant 
to  phagocytosis  when  cultivated  on  immune  sera  or  passed  through  the 
animal  body. 

Thus  the  power  to  invade  depends  possibly  upon  a  combination  of 
offensive  properties  and  defensive  qualities  on  the  part  of  the  bacteria. 
Added  to  this,  some  of  us  believe  that  the  reaction  between  lytic  anti- 
bodies and  the  bacterial  protein  may  produce  toxic  substances  which 
poison  the  animal  body,  prevent  positive  chemotaxis,  and  thereby  aid 
the  invader. 

Again,  there  are  microorganisms  like  the  treponema  pallidum  in 
syphilis  where  adaptation  between  invader  and  host  seems  to  be  of 
such  a  nature  that  an  indifferent  reaction  against  the  invading  organism 
only  is  set  up. 

1  Citrmi,  Cent.  f.  Bakt.,  I,  xl,  1905;  xU,  1906;  and  Zeit.  f.  Hyg.,  Hi,  1905. 

^Zinsser  and  Dwyer,  Proceedings  of  the  Soc.  forExper.  Biol,  and  Med.,  1914,  xi,  74-76, 


CHAPTER  XIX 
ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY 

PHENOMENA   OF   ANAPHYLAXIS 

The  phenomena  now  grouped  together  under  the  heading  of  anaphy- 
laxis and  hypersusceptibiUty  have  but  recently  become  the  subject  of 
systematic  experimentation.  Nevertheless,  manifestations  now  recog- 
nized as  belonging  to  this  category,  had  not  escaped  the  attention  of  a 
number  of  the  earlier  workers  in  immunity. 

By  anaphylaxis  is  meant  the  following  train  of  phenomena:  When 
a  foreign  proteid  is  introduced  by  subcutaneous,  intraperitoneal,  intra- 
venous, or  subdural  injection  (or  in  some  cases  by  feeding)  into  the 
animal  body,  after  a  time  there  will  appear  a  specific  hypersuscepti- 
biUty of  the  animal  for  this  proteid.  After  a  definite  interval,  a  second 
injection  of  the  same  substance,  harmless  in  itself,  will  produce  violent 
symptoms  of  illness  and  often  rapid  death  in  an  animal  so  prepared. 
The  phenomena  are  not  limited  to  any  given  class  of  proteids,  but  are 
manifest  in  the  case  of  animal,  vegetable,  and  bacterial  proteids,  and 
within  certain  limits  are  specific. 

As  early  as  1893,  Behring  ^  and  his  pupils  ^  had  noticed  that  animals, 
highly  immunized  against  diphtheria  toxin,  with  high  antitoxin  content 
of  the  blood,  would  occasionally  show  marked  susceptibility  to  injections 
of  small  doses  of  the  toxin. 

The  phenomena  observed  by  them  was  interpreted  as  an  increased 
tissue  susceptibility  to  the  toxin,  and  Wassermann,  reasoning  on  the 
basis  of  Ehrlich's  side-chain  theory,  formulated  the  conception  that  the 
increased  susceptibihty  was  due  to  toxin  receptors,  increased  in  number 
by  immunization,  but  not  yet  separated  from  the  cells  that  had  produced 
them;  the  cells  thereby  becoming  more  vulnerable  to  the  poison.  In 
the  same  category  belongs  the  observation  of  Kretz,  who  noticed  that 
normal  guinea-pigs  did  not  show  any  reaction  after  injections  of  innocuous 

I  Behring,  Deut.  med.  Woch.,  1893. 

^KnojTf  Dissert.,  Marburg,  1895;  Behring  und  Kitashina,  Berl.  klin.  Woch., 
1901. 


296  INFECTION  AND  IMMUNITY 

toxin-antitoxin  mixtures,  but  that  marked  symptoms  of  illness  often 
followed  such  injections  when  made  into  immunized  guinea-pigs.  Other 
phenomena  which  are  now  regarded,  a  'posteriori,  as  probably  depending 
upon  the  principles  involved  in  anaphylaxis,  are  the  tuberculin  and 
mallein  reactions,  fully  described  in  another  place,  and  the  adverse 
effects  often  following  the  injections  of  antitoxins  in  human  beings, 
conditions  spoken  of  under  the  heading  of  ''serum  sickness."  The  last- 
named  condition  has  been  made  the  subject  of  an  exhaustive  study  by 
v.  Pirquet  and  Schick.* 

That  the  injection  of  diphtheria  antitoxin  in  human  beings  is  often 
followed,  after  an  incubation  time  of  from  three  to  ten  days,  by  ex- 
anthematous  eruptions,  urticaria,  swelling  of  the  lymph  glands,  and 
often  albuminuria  and  mild  pulmonary  inflammations,  has  been  noticed 
by  many  clinicians,  who  have  made  extensive  therapeutic  use  of  anti- 
toxin. It  was  recognized  early  that  such  symptoms  were  entirely  inde- 
pendent of  the  antitoxic  nature  of  the  serum,  but  depended  upon  other 
constituents  or  properties  peculiar  to  the  antitoxic  serum.  Moreover, 
symptoms  of  this  description  were  by  no  means  regular  in  patients  in- 
jected for  the  first  time,  but  seemed  to  depend  upon  an  individual  pre- 
disposition, or  idiosyncrasy,  v.  Pirquet  and  Schick,  however,  noticed 
that  in  those  injected  a  second  time,  after  intervals  of  weeks  or  months, 
the  consequent  evil  effects  were  rapid  in  development,  severe,  and 
occurred  with  greater  regularity.  Many  of  the  phases  of  such  ''serum 
sickness"  are  still  obscure,  since  experimental  conditions  can  not  be 
controlled,  and  many  modifying  factors  can  not  be  excluded  in  observa- 
tions made  upon  human  beings,  and  the  grouping  of  the  above  conditions 
with  the  phenomena  of  anaphylaxis  is  still  tentative. 

The  fundamental  observations  from  which  our  present  knowledge  of 
anaphylaxis  takes  its  origin  are  those  made  in  1898  by  Hericourt  and 
Richet,^  who  observed  that  repeated  injections  of  eel  serum  into  dogs 
gave  rise  to  an  increased  susceptibility  toward  this  substance  instead 
of  immunizing  the  dogs  against  it.  Following  up  the  lines  of  thought 
suggested  by  this  phenomenon,  Portier  and  Richet^  later  made  an  in- 
teresting observation  while  working  with  actino-congestin — a  toxic 
substance  which  they  extracted  from  the  tentacles  of  Actinia.     This 

^v.  Pirquet  and  *Sc/iicA;,  "  Die  Serum  Krankheit,"  monograph,  Leipzig  andWien, 
1905. 

^Hericourt  and  Richet,  Compt.  rend,  de  la  see.  de  biol.,  53,  1898. 

^Portier  and  Richet,  Compt.  rend,  de  la  soc.  de  biol.,  1902;  Richet,  Ann.  de 
I'inst.  Pasteur,  1907  and  1908. 


ANAPHYLAXIS  Otl  HYPERSUSCEPTIBILITY  297 

substance  in  doses  of  0.042  grams  per  kilogram  produced  vomiting, 
diarrhea,  collapse,  and  death  in  dogs.  If  doses  considerably  smaller 
than  this  were  given  in  quantities  sufficient  to  cause  only  temporary 
illness,  and  several  days  allowed  to  elapse,  a  second  injection  of  a 
quantity  less  than  one-quarter  or  one-fifth  of  the  ordinary  lethal  dose 
would  cause  rapid  and  severe  symptoms  and  often  death.  Similar 
observations  were  made  soon  after  this  by  Richet  with  mytilo-conges- 
tin,  a  toxic  substance  isolated  from  mussels.  In  these  experiments  there 
remained  little  doubt  as  to  the  fact  that  the  first  injection  had  given  rise 
to  a  well-marked  increased  susceptibility  of  the  dogs  for  the  poison  used. 

It  was  Richet  who  first  applied  to  this  phenomenon  the  term  "  ana- 
phylaxis" {avd  against,  (poXd^t^  protection),  to  distinguish  it  from 
immunization  or  prophylaxis. 

Soon  after  Richet's  earlier  experiments,  and  simultaneously  with  his 
later  work,  Arthus^  made  an  observation  which  plainly  confirmed 
Richet's  observations,  though  in  a  somewhat  different  field.  The  ob- 
servation of  Arthus  is  universally  spoken  of  as  the  ''phenomenon  of 
Arthus." 

He  noticed  that  the  injection  of  rabbits  with  horse  serum  (a  sub- 
stance in  itself  without  toxic  properties  for  normal  rabbits)  rendered 
the  rabbits  delicately  susceptible  to  a  second  injection  made  after  an 
interval  of  six  or  seven  days.  The  second  injection — even  of  small  .doses 
— regularly  produced  severe  symptoms  and  often  death  in  these  animals. 

An  observation  very  similar  to  that  of  Arthus  was  made  by  Theobald 
Smith  ^  in  1904.  Smith  observed  that  guinea-pigs  injected  with  diph- 
theria toxin-antitoxin  mixtures  in  the  course  of  antitoxin  standardiza- 
tion, would  be  killed  if  after  a  short  interval  they  were  given  a  subcu- 
taneous injection  of  normal  horse  serum. 

The  fundamental  facts  of  hypersusceptibility  had  thus  been  observed, 
and  Otto,^  working  directly  upon  the  basis  of  Smith's  observation, 
carried  on  an  elaborate  inquiry  into  the  phenomenon.  Almost  simul- 
taneously with  Otto's  publication  there  appeared  a  thorough  study  of 
the  condition  by  Rosenau  and  Anderson.* 

The  researches  of  Otto,  and  Rosenau  and  Anderson,  besides  con- 
firming the  observations  of  previous  workers,  brought  out  a  large  number 

1  Arthus,  Compt.  rend,  de  la  soc.  de  bioL,  55,  1903. 

2  Th.  Smith,  Jour.  Med.  Res.,  1904. 

3  Otto,  "Leuthold  Gedenkschrift,"  1905. 

*  Rosenau  and  Anderson,  Hyg.  Lab.  U.  S.  Pub.  Health  and  Marine  Hosp.  Serv. 
Bull.,  29,  36,  1906,  1907. 
20 


29g  INFECTION  AND  IMMUNITY 

of  new  facts.  They  showed  conclusively  that  the  action  of  the  horse 
serum  had  no  relationship  to  its  toxin  or  to  its  antitoxin  constituents, 
that  the  ''sensitization"  of  the  guinea-pigs  by  the  first  injection  became 
most  marked  after  a  definite  incubation  time  of  about  ten  days.  Sen- 
sitization was  accomplished  by  extremely  small  doses  (one  one-millionth 
in  one  case,  usual  doses  sh  to  1  c.c).  Rosenau  and  Anderson,  further- 
more, excluded  hemolysin  or  precipitin  action  as  explanations  of  the 
phenomena,  and  proved  that  hypersusceptibility  was  transmissible  from 
mother  to  offspring,  and  that  it  was  specific — animals  sensitized  with 
horse  serum  not  being  sensitive  to  subsequent  injections  of  other  pro- 
teids.  These  authors,  Vaughan^  and  Wheeler,  Nicolle,^  and  others, 
furthermore,  showed  that  the  reaction  was  by  no  means  limited  to  animal 
sera,  but  was  elicited  by  proteins  in  general,  pepton,  egg  albumin,  milk, 
the  extract  of  peas,  and  bacterial  extracts. 

The  typical  anaphylactic  reaction,  then,  is  obtained  when  animals, 
preferably  guinea-pigs,  are  injected  with  a  small  quantity  of  a  given 
protein,  and  ten  or  fifteen  days  subsequently  given  a  second  injection 
of  the  same  substance  employed  for  the  first  or  sensitizing  inoculation. 
The  quantity  used  for  the  second  injection  should  be  considerably 
larger  than  that  used  for  sensitization  when  the  injection  is  made 
intraperitoneally  or  subcutaneously.  When  given  intravenously,  intra- 
cranially,  or  intracardially,  amounts  as  small  as  0.25  to  0.008  c.c.  may 
suffice.  The  time  at  which  a  second  injection  gives  rise  to  the  most 
violent  symptoms,  moreover,  is  to  a  large  extent  dependent  upon  the 
size  of  the  sensitizing  dose.^  After  extremely  small  initial  quantities 
(0.005-0.002  c.c),  the  anaphylactic  state  is  usually  well  developed,  ac- 
cording to  Rosenau  and  Anderson,^  after  twelve  or  fourteen  days.  After 
larger  doses  ^  the  time  required  for  the  development  of  anaphylaxis  is 
usually  longer — extending  often  over  weeks,  or  even  months. 

While  the  sensitizing  or  first  dose  may  be  given  subcutaneously, 
intravenously,  intraperitoneally,  or  intracardially  with  equal  success, 
Besredka  and  Steinhardt  maintain  that  no  anaphylaxis  results  if  the 
first  dose  is  given  intracranially.  This  statement,  however,  has  found 
contradiction  in  the  work  of  Rosenau  and  Anderson.  The  time  required 
for  full  sensitization,  furthermore,  depends,  according  to  the  last-named 
authors,  also  upon  the  mode  of  injection  of  the  first  dose;  on  this  point, 
however,  no  conclusions  are,  at  present,  justified. 

1  Vaughan,  Assn.  Am.  Phys.,  May,  1907.    ^  Besredka,  Ann.  de  I'inst.  Pasteur,  1907. 

2  Nicolle,  Ann.  de  1'  Inst.  Pasteur,  2,  1903.      ,       •*  Rosenau  and  Anderson,  loc.  cit. 
6  Otto,  Munch,  med.  Woch.,  1907. 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY  299 

At  reinjection,  the  symptoms  are  most  severe  when  the  injection  is 
made  intravenously. 

The  symptoms  which  follow  on  the  reinjection  of  antigen  into  sensitive 
animals  may  show  a  wide  range  of  variation  according  to  the  degree  of  sensi- 
tiveness and  amounts  injected.  In  acute  anaphylaxis  of  guinea-pigs,  which  aa 
you  know  has  been  the  most  thoroughly  studied,  there  is  a  rapid  and  severe 
death  which  may  not  occupy  more  than  a  fraction  of  a  minute  or  at  most  five 
to  ten  minutes.  The  animals  repeatedly  show  restlessness,  cough,  pass  urine 
and  feces,  develop  severe  dyspnea,  with  infrequent  respiration  in  which  there 
seems  to  be  almost  complete  inamobiUzation  of  the  chest  wall  and  in  which 
finally  only  shallow,  irregular,  spasmodic  efforts  take  place.  This,  as  Auer  and 
Lewis  have  shown,  is  due  to  tetanic  contraction  of  the  small  bronchioles,  with 
occlusion  of  the  air  passages,  practically  no  air  entering  the  lungs.  As  the 
dyspnea  develops,  there  may  be  at  the  same  time  spasmodic  twitching  of  the 
limbs,  retraction  of  the  head  and  general  convulsions. 

When  for  some  reason  or  other  the  reaction  is  not  so  severe  the  animal 
may  show  merely  general  signs  of  illness,  ruffhng  of  the  fur,  twitching  and 
restlessness,  with  respiratory  diflBiculty  of  varying  degree,  coughing,  and  evacua- 
tion of  urine  and  feces.  In  rabbits  the  symptoms  are  often  less  rapid  in  de- 
velopment, but  in  general  principles  are  similar;  in  rabbits  there  is  more  fre- 
quently in  the  moderate  cases  a  gradual  muscular  weakness  in  which  the  an- 
imal lies  flat  on  the  ground  unable  to  support  itself  on  its  legs,  a  condition 
which  may  proceed  for  long  periods.  Death  is  largely  respiratory  and  the 
heart  may  continue  to  beat  for  a  long  time  after  respiration  has  completely 
stopped.    There  is  a  sinking  of  blood  pressure  and  a  depression  of  temperature. 

The  coagulation  time  of  the  blood  is  lengthened,  there  is  apparently  a  depres- 
sion of  the  leukocytes,  and  according  to  a  number  of  investigators,  who  have 
been  recently  confirmed  by  Behring,  there  is  a  disappearance  of  blood  platelets 
and  an  increased  flow  of  lymph. 

Pathologically  in  an  animal  dead  of.  anaphylaxis  there  may  be  petechial 
hemorrhages,  according  to  Gay  and  Southard,  in  the  heart  muscle,  pleura  and 
intestinal  waU  and  there  may  be  fatty  degeneration  of  the  vascular  endothehum. 
In  guinea-pigs  especially  there  is  a  marked  emphysematous  dilatation  of  the 
4imgs  which  is  very  constant,  although  according  to  Doerr  it  is  not  absolutely 
characteristic  of  this  condition.  Apart  from  the  anatomical  changes  following 
acute  anaphylaxis,  frequently  repeated  injections  of  small  doses  of  horse  serum 
or  egg  white  in  dogs,  cats,  rabbits  and  guinea-pigs  have  been  shown  by  Long- 
cope  to  produce  cell  injury  in  various  organs,  especially  in  the  hver,  myo- 
cardium and  kidneys. 

When  sensitized  animals  recover  from  the  second  injections,  they 
are  thereafter  immune — that  is,  they  do  not  react  to  subsequent  in- 
jections of  the  same  substance. 


300      .  INFECTION  AND  IMMUNITY 

This  desensitization  or  ^'antianaphylaxis"  as  Besredka  ^  and  Stein- 
hardt  have  called  it,  appears  immediately  after  recovery  from  the  second 
injection.  Antianaphylaxis  may  also  be  produced  if  animals  which  have 
receiv^ed  the  first  or  sensitizing  dose  are  injected  with  comparatively 
large  quantities  of  the  same  substance  during  the  preanaphylactic  period 
— or,  as  it  is  sometimes  spoken  of,  during  the  anaphylactic  incubation 
time.  This  injection  should  not  be  done  too  soon  after  the  first  dose,  but 
rather  toward  the  middle  or  end  of  the  preanaphylactic  period. 

If  given  within  one  or  two  days  after  the  sensitizing  injection,  ana- 
phylaxis will  develop,  nevertheless.  The  desensitized  condition  is  a 
purely  transitory  state.  Besredka  and  Steinhardt  believe  that  it  lasts 
a  long  time,  while  Otto  found  guinea-pigs  immunized  in  the  above  man- 
ner to  lose  their  antianaphylaxis  within  three  weeks. 

An  important  development  was  achieved  when  Nicolle,  Otto,^  Gay 
and  Southard,^  and  others  *  showed  that  the  hypersusceptible  state 
could  be  passively  transferred  to  normal  animals  by  injecting  them  with 
the  serum  of  anaphylactic  animals.  In  such  experiments  the  serum  of 
the  anaphylactic  animal  is  first  injected  in  quantities  of  0.5  c.c.  or  prefer- 
ably more,  and  twenty-four  hours  later  an  injection  of  the  specific  anti- 
gen— -that  is,  the  proteid  used  for  sensitization — is  given.  The  animals 
so  treated  show  typical  symptoms  of  hypersusceptibility  and  often  die. 

Simultaneous  inoculation  of  the  two  substances,  either  mixed  or  in- 
jected separately,  does  not  produce  the  same  effect.  On  this  point, 
however,  there  is  not  complete  unanimity,  since  Weill-Halle  and  Le- 
maire  ^  report  aphylactic  symptoms  in  guinea-pigs  hypersusceptible  to 
horse  serum.  A  fact,  observed  by  Otto,  is  that  the  serum  of  guinea-pigs 
who  have  been  given  the  sensitizing  or  first  injection  will  confer  passive 
anaphylaxis  on  the  eighth  or  tenth  day  after  injection,  before  the  ani- 
mals themselves  show  evidences  of  being  actively  hypersensitized.  It 
is  also  true  that  occasionally  the  serum  of  antianaphylactic  animals  will 
possess  the  power  of  conferring  passive  anaphylaxis. 

Anaphylaxis  may  be  transmitted  by  inheritance.  Thus  the  young 
of  anaphylactic  guinea-pigs  show  hypersusceptibility,  irrespective  of 
whether  the  mother  became  hypersusceptible  before  or  after  the  be- 
ginning of  pregnancy.  Such  anaphylaxis  has  no  reference  to  the  con- 
dition of  the  father,  and  is  not  transmitted  by  the  milk. 

1  Besredka  and  Steinhardt,  Ann.  de  ITnst.  Pasteur,  1907. 

2  Otto,  loc.  cit.  ^Gay  and  Southard,  loc.  cit. 
*  Weill-Halle  and  Lemaire,  Compt.  rend,  de  la  Soc.  de  Biol,,  1907. 

^NicoUe,  Ann.  de  ITnst.  Pasteur,  1907,  1908. 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY  301 

THEORIES  CONCERNING  ANAPHYLAXIS 

A  number  of  theories  of  anaphylaxis  have  been  advanced  which  de- 
serve serious  consideration,  since  they  are  experimentally  supported 
and  may  serve  as  points  of  departure  for  future  research. 

One' of  the  earliest  ideas  was  based  on  the  Ehrlich  theory  of  re- 
ceptor overproduction  by  tissue  cells  during  immunization.  It  was 
suggested  that  hypersusceptibility  might  be  due  to  stimulation  of  new 
specific  receptors  which  as  yet  remained  sessile  upon  the  cells  instead 
of  having  been  thrown  off  into  the  blood  stream.  As  a  consequence, 
the  cells,  having  affinity  for  more  of  the  toxic  substance  of  the  antigen 
than  they  had  normally,  are  more  vulnerable. 

V.  Pirquet  and  Schick,^  as  well  as  many  other  observers,  have  re- 
garded the  anaphylactic  process  as  analogous  to  other  immune  reactions, 
and  believe  that  an  antigen  in  the  serum  first  injected  produces  a  specific 
antibody.  The  reaction  between  these  two  substances  following  the 
second  injection  gives  rise  to  the  anaphylactic  symptoms.  The  essen- 
tial feature  of  this  opinion  is  the  assumption  that  the  substance  which 
sensitizes  after  the  first  injection  is  identical  with  that  which  exerts  the 
anaphylactic  injury  after  reinjection. 

Wolff-Eisner  ^  has  expressed  a  belief  which  has  found  much  experi- 
mental support  in  the  hands  of  Vaughan  and  Wheeler.^  Wolff-Eisner 
holds  that  all  cells  and  proteins  contain  a  toxic  substance  which  is 
characterized  by  its  inability  to  produce  a  neutralizing  antibody  when 
injected  into  animals.  The  first  injection  produces  a  lysin  for  the  pro- 
teid  injected,  which  possesses  the  power  of  liberating  such  poisons  from 
the  complex  molecule.  A  second  injection  is  followed,  consequently, 
by  a  rapid  liberation  of  the  toxic  fraction,  and  injury  to  the  animal 
results.  This  view  has  been  expressed  in  slightly  different  form  by 
Richet  ^  and  has  been  more  clearly  formulated  and  experimentally  sup- 
ported by  Vaughan  and  Wheeler  "^  who  were  actually  able  to  extract 
from  various  proteins  toxic  substances  which  gave  rise  in  animals  to  a 
symptom  complex  not  unlike  that  of  typical  anaphylaxis.  (Extraction 
with  alkalinized  seventy-per-cent  alcohol.) 

The  earlier  theories  of  Gay  and  Southard  ^  and  that  of  Besredka  ^ 

1  V.  Pirquet  und  Schick,  loc.  cit.  2  Wolff-Eisner,  Berl.  klin.  Woch.,  1904. 

^Richet,  Ann.  de  I'lnst.  Pasteur,  xxi,  1907. 

*  Vaughan  and  Wheeler,  Jour.  Infect.  Dis.,  iv,  1907. 

^  Gay  and  Southard,  Jour.  Med.  Research,  1907,  xvi. 

6  Besredka,  Bull,  de  I'lnst.  Pasteur,  1908,  vi,  826. 


302  INFECTION  AND  IMMUNITY 

we  may  at  present  dismiss  with  a  few  words  as  interesting  developments 
in  the  theoretical  study  of  anaphylaxis  but,  since  then,  proved  to  be 
of  little  importance  in  the  actual  understanding  of  the  phenomena. 
Many  of  these  earlier  theories  were  made  possible  by  lack  of  sufficient 
evidence  that  anaphylaxis  depended  upon  the  reaction  between  an  anti- 
body and  its  antigen  and  upon  attempts  to  show  that  the  sul)stance 
which  was  sensitized  was  not  the  same  as  that  which  produced  the  toxic 
symptoms  on  reinjection.  This  last  opinion  was  mainly  based  upon 
such  experiments  as  those  of  Besredka  which  seemed  to  show  that  the 
toxogenic  was  less  heat  stable  than  the  sensitizing  substance,  but  since 
the  amount  which  sensitizes  need  be  only  anywhere  from  1-lOOth  to 
1-lOOOth  part  of  the  amount  required  for  the  production  of  toxic  symp- 
toms, as  Wells  and  others  have  calculated,  it  is  plain  that  this  was  the 
explanation  for  the  apparent  difference  in  heat  stability  between  the 
two.  Chemical  differentiation  between  the  two  substances,  as  attempted 
by  Gay  and  Adler,  has  likewise  failed  to  hold  good.  We  have  now  come 
to  the  recognition  that  anaphylaxis  is  the  result  of  the  presence  of  spe- 
cific antibodies  in  the  animal  into  which  the  antigen  is  injected.  These 
antibodies  can  be  produced  on  the  one  hand  by  a  preliminary  injection 
of  the  antigen,  thus  stimulating  the  animal  to  produce  its  own  anti- 
bodies, or  by  the  direct  injection  of  the  antibodies  from  another  animal 
as  in  passive  sensitization  with  immune  serum. 

The  development  of  antibodies  in  the  animal  then  alters  the  reaction 
capacity  of  the  animal  for  the  particular  antigen  employed.  This  is 
the  state  which  v.  Pirquet  has  named  "allergic,"  and  it  is  this  allergie 
or  changed  reaction-capacity  due  to  the  formation  of  antibodies,  whether 
circulating  or,  as  is  more  probable,  attached  to  the  cells  themselves, 
which  induces  in  the  animal  the  anaphylactic  state. 

It  is  by  means  of  the  passive  method  of  sensitization  that  the  relations 
between  anaphylaxis  and  antibodies  have  been  most  successfully  stud- 
ied. Doerr  and  Russ  ^  showed  that  the  power  of  a  serum  to  convey 
anaphylaxis  passively  depended  directly  upon  its  contents  of  specific 
antibody.  It  was  then  shown  by  Nicolle,^  Otto,^  and  others,  that  a 
sharp  reaction  can  be  produced  by  this  method  only  when  a  distinct 
interval  not  less  than  4  to  6  hours,  was  allowed  to  lapse  between  the 
injection  of  the  antibodies  and  the  injection  of  the  antigen.    This  may 

1  Doerr  and  Russ,  Ztschr.  f.  Immunitatsforsch.,  1909,  iii. 

2  Nicolle,  Bull,  de  I'lnst.  Past.,  1907,  v. 

3  Otto,  Das  Theobald  Smithsche  Phaenomenon,  etc.,  von  Leuthold  Gedenkschrift 
1905,1. 


ANAPHYLAXIS  OR  HYPERSUSCEPTIBILITY  303 

be  taken  as  an  axiom  for  all  cases  in  which  the  antigen  is  an  unformed 
protein  in  solution.  Whether  or  not  it  holds  universally  good  in  the 
case  of  cell  anaphylaxis  is  a  question  too  complicated  to  be  discussed 
here.  When  the  antigen  and  antibody  are  injected  simultaneously  or 
within  a  very  short  period  of  one  another,  no  anaphylactic  symptoms 
occur.  The  study  of  this  interval  has  gradually  led  to  the  belief  that 
the  anaphylactic  reaction,  whatever  it  may  be,  takes  place  upon  the 
body  cells  and  that  the  interval  is  necessitated  by  the  time  required  for 
the  anchoring  of  the  antibodies  to  the  cells  of  the  body.  Experiments 
by  Pearce  and  Eisenbrey^  (1910)  showed  definitely  that  a  sensitized 
dog  remained  sensitized  even  when  his  entire  blood  volume  is  substi- 
tuted with  that  of  a  normal  dog.  It  has  been  made  especially  clear 
by  the  introduction  of  direct  methods  of  observation  of  the  smooth 
muscle  cells  of  animals  by  Schultz^  and  Dale,^  a  method  which  has 
been  particularly  developed  by  Weil.^  It  seems  fairly  clear  from  this 
work  and  a  volume  of  other  researches  which  cannot  be  reviewed  here, 
that  acute  protein  anaphylaxis  as  we  see  it  in  guinea-pigs  and  other 
laboratory  animals  is  due  to  the  direct  reaction  between  antigen  arid 
a  specific  antibody  when  this  reaction  takes  place  upon  the  body  cells 
and  not  in  the  blood  stream.  Just  how  much  influence  the  reaction 
within  the  blood  steam  can  exert  or  whether  it  takes  any  important  part 
in  the  phenomena  is  at  present  a  matter  of  considerable  doubt. 

An  important  observation  made  by  Friedberger  and  Hartoch  ^  and 
much  studied  by  others  recently,  also,  has  been  the  diminution  of  com- 
plement or  alexin  in  the  serum  of  animals  suffering  from  anaphylactic 
shock.  It  has  been  shown  that  in  passive  anaphylaxis,  at  least  simul- 
taneously with  the  occurrence  of  symptoms,  there  is  a  marked  diminu- 
tion of  alexin.  Intravenous  injections  of  substances  which  prevented 
complement  absorption  in  vitro  (concentrated  salt  solution,  for  in- 
stance) diminished  shock  and  sometimes  prevented  it  in  sensitized 
animals.  They  attributed  this  to  the  fact  that  complement  was  thereby 
inhibited,  and  concluded  that  complement  took  an  important  part  in 
the  reaction.  Incidentally  it  may  be  mentioned  that  in  experiments 
upon  the  uterine  contractions  of  sensitized  guinea  pigs,  the  writer  with 

1  Pearce  and  Eisenbrey,  Congr.  Am.  Phys.  and  Surg.,  1910,  viii. 

2  Schultz,  Jour.  Pharmacol,  and  Exper.  Therap.,  1910,  i. 

3  Dale,  Jour.  Pharmacol,  and  Exper.  Therap.,  1913,  iv. 

4  Weil,  Jour.  Med.  Research,  27,  1913;  30,  1914;  Proc.  Soc.  Exper.  Biol,  and  Med., 
1914,  xi,  86. 

^  Friedberger  and  Hartoch,  Ztschr.  f,  Immunitatsforsch.,  1909,  iii. 


304  INFECTION  AND  IMMUNITY 

Lieb  and  Dwyer  ^  has  been  able  to  show  that  action  of  the  salt  pre- 
vents spasm  of  the  smooth  muscles  and  need  not  necessarily  be  inter- 
preted as  indicating  an  inhibition  of  complement  action. 

An  important  development  of  Friedberger's  work  has  been  the  dem- 
onstration that  when  sensitized  antigen,  either  in  the  form  of  precipitate 
or  of  bacteria,  is  subjected  in  vitro  to  the  action  of  alexin  or  complement, 
toxic  substances  (anaphylatoxins)  are  formed,  which  injected  into 
guinea-pigs  produce  symptoms  entirely  analogous  to  anaphylactic 
shock.  This  has  led  to  a  recognition  of  the  great  importance  of 
Vaughan's  earlier  work  in  pointing  to  the  possibility  of  anaphylactic 
shock  being  the  result  of  poisons  formed  by  the  split  products  of  pro- 
teins. Whether  or  not  these  anaphylatoxins  are  actually  produced  in 
the  circulation  of  animals  in  the  process  of  anaphylactic  shock  cannot 
be  definitely  asserted  at  present.  It  seems  rather  unlikely  that  they 
are  produced  in  the  circulation  to  any  extent  during  shock  produced 
by  the  dissolved  proteins  such  as  foreign  serum  or  egg  albumin.  It  is 
not  impossible  that  they  may  be  formed  in  the  course  of  cell  anaphylaxis 
and  that  they  may  play  a  considerable  part  in  the  toxemia  accompanying 
infectious  disease.  In  fact,  their  universal  production  from  practically 
all  known  bacteria,  and  the  experiments  of  Vaughan  ^  which  have  shown 
that  similar  poisons  produced  from  proteins  can  lead  to  typical  tempera- 
ture reactions  in  animals  if  carefully  and  systematically  injected,  has 
cast  a  certain  amount  of  doubt  upon  the  existence  of  true  endotoxins 
and  has  suggested  to  many  workers  the  possibility  that  the  toxemia 
produced  with  such  bacteria  as  typhoid  bacillus  and  others  may  be 
largely  a  protein  split  product  intoxication  or  a  sort  of  prolonged  ana- 
phylactic poisoning. 

It  seems  unquestionable  that  this  altered  state  of  reaction  capacity 
to  specific  antigens  which  v.  Pirquet  has  called  allergic,  and  which  we 
speak  of  broadly  as  anaphylaxis,  underlies  many  conditions  observed 
in  the  human  being.  Most  important  among  these  is  the  frequently 
observed  serum  disease  which  occurs  after  the  injection  of  horse  serum 
and  other  sera  in  the  form  of  antitoxins  in  the  human  being.  It  may 
also  confirm  the  pathological  basis  of  such  conditions  as  asthma  and 
hay  fever,  food  idiosyncrasies,  and  in  a  localized  way  it  is  probably 
the  basis  of  such  skin  reactions  as  the  tuberculin,  luetin,  and  mallein 
reaction. 

1  Zinsser,  Ideb,  and  Dwyer,  Proc.  Soc.  Exper.  Biol,  and  Med.,  Vol.  XII,  No.  8, 
May  19,  1915. 

2  Vaughaui  Protein.  SpUt.  Products,  etc..  Lea  and  Febiger,  1913., 


CHAPTER  XX 

FACTS  AND    PROBLEMS    OF  IMMUNITY    IN    THEIR  BEARING  UPON 
THE  TREATMENT  OF  INFECTIOUS  DISEASES 

While  the  various  facts  and  theories  of  immunity  and  infection  have 
been  given  in  the  preceding  sections,  no  systematic  attempts  have  been 
made  to  correlate  the  facts  presented,  or  to  determine  their  bearing  on 
the  most  vital  problem  of  all — the  treatment  of  infectious  diseases. 

To  understand  more  fully  this  point  of  view,  it  is  necessary  briefly 
to  recall  certain  of  the  facts  which  are  known  about  the  physiology, 
metabolism,  and  composition  of  the  bacteria,  and  of  their  ability  to 
neutralize  directly  or  to  respond  adaptively  to  the  agents  directed  against 
them  by  the  invaded  animal.  Some  of  these  facts  are  so  well  understood 
that  passing  mention  here  is  sufficient:  such,  for  instance,  is  the  fact 
that  certain  microorganisms,  especially  the  bacilli  of  diphtheria  and  tet- 
anus, secrete  soluble  poisons  both  during  artificial  cultivation  and  dur- 
ing their  life  in  the  animal  body,  which  poisons  are  eminently  toxic. 
These  poisons  are  true  secretions  and  are  largely  independent  of  the 
composition  of  the  surrounding  medium  so  long  as  this  favors  the  physi- 
ologic activities  and  growth  of  the  germs.  Such  germs,  then,  once  having 
gained  even  an  insecure  foothold  in  the  animal  body,  by  no  matter  w^hat 
favoring  circumstances,  are  possessed  of  a  powerful  weapon  of  offense 
against  the  sensitive  physiologic  bases  of  the  host  and,  possibly,  of  de- 
fense  against  its  more  immediate  and  mobile  means  of  combating  the 
germs  themselves.  In  the  case,  however,  of  most  other  pathogenic 
bacteria,  the  secretion,  at  least nn  artificial  media,  of  such  highly  soluble 
and  potent  poisons  has  not  been  demonstrated  satisfactorily,  although 
certain  investigations  point  fairly  conclusively  to  the  production  of 
some  minor  bodies  which  have  been  shown  to  act  deleteriously  on  the  red 
blood  cells  and  on  the  leucocytes — the  hemolytic,  leucocidic,  and  leu- 
colytic  substances  which  are  looked  on  as  probably  true  soluble  toxins, 
like  the  toxins  of  diphtheria  and  tetanus,  which  give  rise  in  the  animal 
body  to  the  production  of  true  antitoxins:  i.e.j  are  neutralized  by 
their  antisera,  uftit  for  unit,  according  tp.  th^.  l^-w;  of  multiples., 

205. 


306  ,  INFECTION  AND  IMMUNITY 

Other  minor  poisons  may  in  some  instances  be  demonstrated  in 
culture  media,  and  also  may  possibly  be  formed  in  the  animal  body  by 
the  metabolic  activities  of  the  germs.  These  are  either  simply  waste 
products  of  metabolism  or  bodies  due  to  the  decomposition  of  the 
nutrient  media  in  which  the  germs  are  gowing.  These  bodies  are  usually 
referred  to  as  ptomains,  and  differ  entirely  from  the  true  secreted  toxins, 
both  in  their  chemical  composition  and  in  their  physiologic  action,  re- 
sembHng  in  both  of  these  the  alkaloids.  They  are  not  known  to  give 
rise  to  antibodies  of  any  kind  in  animals. 

Apart  from  all  the  poisons  just  mentioned;  i.e.,  the  toxins,  hemoly- 
sins, leucocidins,  and  ptomains,  there  is  supposed  to  exist  a  most  vitally 
important  and  interesting  group  of  poisonous  substances,  the  so-called 
endotoxins.  These,  so  far  as  our  knowledge  goes,  are  poisons  rather 
firmly  seated  in  the  bacterial  cell,  which  are  not  secreted  in  our  ordinary 
cultural  media,  and  are  supposed  by  most  observers  not  to  be  separable 
in  the  animal  fluids  and  tissues  from  the  intact  bacterial  cell.  These 
poisons  may  be  demonstrated  in  old  cultures,  in  which  the  bacteria 
are  dead  and  disintegrating  or  undergoing  autolysis — although  Pfeiffer 
does  not  consider  autolytic  products  necessarily  similar  to  endotoxins 
— or  they  may  be  obtained  by  destroying  the  bacteria  mechanically  by 
pressure  and  grinding,  or  by  breaking  them  while  frozen.  In  the  animal 
body  they  are  said  to  become  free  when  the  bacteria  die  and  decompose 
or  are  disintegrated  by  the  digestive  bodies  by  which  they  have  been 
attacked.  These  endotoxins  are  recognized  by  the  fact  that  they 
do  not  call  out  true  antitoxins  which  become  free  in  the  plasma  and 
serum,  but  do,  nevertheless,  lead  to  the  formation  of  digestive  antibodies, 
these  not  following,  however,  the  "law  of  multiples"  in  protecting  in- 
fected animals  from  the  poisons.  The  liberation  of  these  poisons  by  the 
destruction  of  bacteria  in  the  animal  body  is  best  illustrated  by  the  so- 
called  phenomenon  of  Pfeiffer  which  takes  place  when  cholera  vibrios 
and  immune  cholera  serum  are  introduced  into  the  peritoneal  cavity 
of  a  guinea-pig.  If  specimens  are  withdrawn  from  time  to  time  from 
the  peritoneal  cavity  of  an  animal  so  treated,  a  rapid  swelling  up, 
disintegration,  and  disappearance  of  the  vibrios  can  readily  be  demon- 
strated. The  organisms  apparently  do  not  multiply  in  the  animal  body 
under  these  conditions  and  are  almost  immediately  destroyed.  This 
disintegrating  power  is  also  claimed  for  the  body  fluids  of  normal 
animals  and  is  supposed  to  be  demonstrated  by  the  following  experi- 
ment. When  graded  quantities  of  a  fresh  cholera  culture  are  introduced 
into  the  peritoneal  cavity  of  normal  gainea-pigs  of  equal   weight,  the 


FACTS  AND  PROBLEMS  OF  IMMUNITY  30? 

following  phenomena  can  be  regularly  observed:  Minimal  doses  of  the 
culture  produce  a  febrile  condition  which  continues  for  a  few  hours  with 
no  serious  symptoms.  Slightly  larger  doses  give  rise,  after  a  short 
interval,  during  which  there  is  fever,  to  a  marked  drop  in  tempera- 
ture and  definite  symptoms  of  cholera  poisoning — muscular  weakness, 
twitching,  and  general  prostration.  These  symptoms  of  poisoning  then 
gradually  disappear,  and  after  twenty-four  hours  the  guinea-pigs  are 
again  normal.  If  the  quantity  of  cholera  culture  injected  is  carefully 
increased  up  to  the  minimal  lethal  dose,  the  animal  dies  w^ith  all  the 
symptoms  of  cholera  intoxication,  but  on  autopsy  the  peritoneum  is 
found  to  be  entirely  sterile,  or  only  a  few  isolated  cholera  spirilla  are 
found,  usually  inclosed  in  pus  cells.  Finally,  if  larger  quantities  of 
living  cholera  spirilla  are  injected,  the  peritoneal  cavity  shows  a  profuse, 
serous,  sometimes  hemorrhagic  exudate,  which  contains  innumerable 
actively  motile  microorganisms.  The  point  of  interest  in  this  experi- 
ment is  the  demonstration  of  the  fact  that  the  normal  guinea-pigs 
which  receive  enough  of  the  cholera  vibrios  to  prove  fatal  have  de- 
stroyed the  vibrios  and  presumably  died  from  the  poison  thus  liberated, 
xnd  not  from  poisons  secreted  by  living  vibrios,  or  from  an  overcoming 
of  their  systems  by  the  rapid  multiplication  of  the  organisms.  It  is  only 
when  the  animal  system  is  previously  flooded  with  an  overwhelming  dose 
that  the  vibrios  are  found  alive  and  multiplying  even  locally  in  the  peri- 
toneum after  death.  This  does  not  mean,  however,  that  no  multiplica- 
cation  ever  goes  on  hand  in  hand  with  the  destruction  of  the  germs  in  the 
infected  animal;  on  the  contrary,  such  a  multiplication  is  probably  the' 
rule  rather  than  the  exception,  as  has  been  shown  fairly  conclusively 
by  the  experiments  of  Radziewsky,  and  was  beautifully  illustrated  by 
an  experiment  of  Pfeiffer  and  Wassermann,  who  after  having  shown  that 
the  blood  serum  of  human  beings  who  have  recovered  from  Asiatic  chol- 
era has  the  power  to  protect  guinea-pigs  from  ordinarily  fatal  doses 
of  cholera  spirilla,  even  when  used  in  high  dilutions,  then  proved  that 
this  protective  power  is  not  an  antitoxic  one,  but  depends  largely,  if 
not  entirely,  on  the  ability  of  the  serum  to  aid  in  the  immediate  dissolu- 
tion of  the  vibrios.  Thus  animals  which  received  only  a  fraction  of  a 
milligram  of  such  a  serum  were  able  to  bear  the  injection  of  a  loopful  of 
virulent  cholera  vibrios,  practically  without  reaction,  while  control 
animals  succumbed  to  one-fourth  of  the  dose  with  typical  symptoms. 
Now,  however,  if  the  dose  was  increased  to  three  or  five  loopfuls,  not 
even  ten  thousand  times  the  original  amount  of  the  serum  would  protect 
the  animals  against  the  inoculation.    The  toxic  effects  may,  in  fact,  as 


308  INFECTION  AND  IMMUNITY 

shown  by  Pfeiffer,  appear  with  extraordinary  rapidity,  so  that  in  these 
animals  the  temperature  may  show  the  lethal  drop  within  two  hours 
after  inoculation,  while  control  animals  which  have  received  the 
same  quantity  of  cholera  germs  without  the  serum  may  not  show  a 
similar  lethal  drop  in  temperature  for  four  to  five  hours. 

An  explanation  of  the  results  of  this  experiment  is  found,  probably, 
in  the  fact  that  guinea-pigs  are  able  to  withstand  a  certain  quantity  of 
the  intracellular  cholera  poison  (endotoxin)  which  may  be  represented  by 
one  loopful  of  a  fresh  culture.  If  the  animals  are  given  smaller  quantities*, 
without  the  serum,  say  one-fourth  to  one-half  loopful,  the  bacteria  may 
increase  for  a  time  without  producing  marked  symptoms.  Parallel' 
with  the  increase,  however,  the  phenomenon  of  germ  destruction  is^ 
going  on  and  characteristic  symptoms  of  intoxication  appear  at  the- 
moment  when  the  number  of  vibrios  destroyed  has  become  so  large  that, 
it  corresponds  to  more  than  one  loopful  of  the  cholera  culture.  An 
animal  will  thus  withstand  a  culture  of  any  size  when  mixed  with  im- 
mune serum,  if  the  dose  does  not  exceed  the  limit  of  intoxication  before 
it  is  entirely  destroyed.  On  the  other  hand,  when  guinea-pigs  receive 
the  larger  dose  of  three  to  five  loopfuls,  the  serum,  not  being  anti- 
toxic, is  not  able  to  counteract  the  fatal  effects  of  the  liberated 
cholera  poisons,  but,  on  the  other  hand,  enormously  increases  the  rate 
of  destruction  of  the  vibrios,  and.  hence  intoxication  appears  earlier 
in  such  treated  animals  than  in  the  controls  receiving  the  organisms 
alone. 

This  classic  cholera  experiment  has  been  selected  because  it  illus- 
trates the  most  extreme  limit  of  the  endotoxin  point  of  view,  and,  further, 
because  the  cholera  organism,  standing  at  one  end  of  the  scale,  is  the 
most  extreme  example  of  pathogenicity  by  virtue  of  its  own  destruction, 
while  the  diphtheria  bacillus  at  the  other  end,  as  we  have  seen,  is  one 
of  the  classic  examples  of  pathogenicity  by  virtue  of  secreted  toxins. 
Neither  of  these  organisms  is  truly  invasive  or  highly  parasitic,  and  both 
are  harmful  usually  by  the  action  of  their  poisons  alone  and  acting,  as  it 
were„  from  a  base  of  supply  on  the  periphery  of  the  animal  system.  Be- 
tween these  two  extremes  stand  all  grades  of  pathogenic  and  infective 
germs. 

.  These  two  organisms  are  typical  examples  of  their  kind,  but  there 
are  few  organisms  which  secrete  such  highly  toxic  soluble  bodies  as  do 
diphtheria  bacilli,  and  there  are  few  so  susceptible  as  the  cholera  organ- 
ism to  disintegra,tion  within  the  animal  body;  and  yet  there  are  many 
germs  which  are  extremely  pathogenic,  and  in  many  cases  capable  of 


FACTS  AND  PROBLEMS  OF  IMMUNITY  309 

severely  and  detrimentally  infecting  the  animal  body.  In  view  of  this 
unquestioned  fact,  the  teaching  which  considers  all  poisonings  as  due 
either  to  true  soluble  secreted  poisons,  or  to  true  endotoxins  Uberated 
only  on  disintegration  of  the  bacterial  cell,  is  probably  too  narrow. 

The  work  of  recent  years  has  even  cast  some  doubt  upon  the 
existence  of  preformed  endotoxins  in  the  bacterial  cell  and  has  indi- 
cated that  the  bacterial  protein  as  such  may  be  regarded  merely  as  a 
foreign  protein  which  would  do  no  special  harm  unless  it  reacted 
with  substances  in  the  animal  body  which  brought  about  its  disinte- 
gration. In  such  a  case  the  toxic  substances  formed  would  not  rep- 
^resent  preformed  poisons,  integral  parts  of  the  bacterial  body,  but 
rather  protein  split  products  derived  from  the  bacteria  by  proteo- 
lytic action  on  the  part  of  the  plasma  constituents. 

Some  clarity  of  conception  may,  as  we  have  suggested,  be  gained 
by  comparing  some  of  the  products  of  pathogenic  bacteria  with  bacterial 
pigments  and  with  insoluble  interstitial  or  intercellular  substance, 
which  may  be  seen, accompanying  bacteria  in  cover-glass  preparations. 
Soluble  toxic  secretions  are  to  be  compared  to  such  pigments  as  the 
pyocyanin  of  Bacillus  pyocaneus,  which  is  so  readily  soluble  in  culture 
media;  endotoxins  proper,  to  pigments  confined  to  the  bacterial  cell, 
or,  at  least  when  secreted,  being  insoluble  in  culture  media,  such,  for 
instance,  as  the  well-known  red  pigment  of  Bacillus  prodigiosus,  which 
may  often  be  seen  free  among  the  bacteria  in  irregular  red  granules 
like  carmine  powder.  That  bodies  such  as  this  latter  might  be  ex- 
truded from  pathogenic  bacteria,  and  not  be  soluble  in  the  usual  culture 
fluids,  is  not  improbable,  and  the  fact  that  more  or  less  insoluble  inter- 
stitial substances  are  not  infrequent  among  bacteria  is  well  known. 
Among  pathogenic  germs  these  characters  are  often  more  marked  in 
freshly  isolated  cultures.  The  sticky,  almost  slimy  character  of  cul- 
tures of  meningococcus  may  be  recalled,  a  character  which  tends  to 
disappear  after  a  few  generations  of  artificial  cultivation,  and  the  highly 
mucinous  capsule  of  the  Streptococcus  mu^osus  which  tends  to  decrease 
under  artificial  cultivation,  as  do  also  the  capsules  of  pneumococci  and 
streptococci. 

Now,  it  seems — and  this  view  has  been  supported  by  Walker, 
Deutsch,  Welch,  and  Eisenberg,  and  is,  in  fact,  but  an  axiom  which 
would  be  recognized  immediately  by  any  trained  biologist — that  all 
microorganisms  will  adapt  themselves  so  far  as  is  permitted  by  their 
physiologic  peculiarities  to  the  stress  of  the  environment,  the  exact 
direction  which  this  adaptation  will  take  being  determined  by  the  char- 


310  INFECTION  AND  IMMUNITY 

acter  of  the  environment,  chemical  and  physical,  and  the  physical, 
chemical,  and  physiologic  characteristics  of  the  germ  involved. 

Having  now  considered  the  bacteria  in  their  defensive  and  offensive 
mechanism,  let  us  turn  to  the  mechanism  of  protection  at  the  disposal 
of  the  animal  body. 

The  internal  defenses  of  the  animal  body  have  largely  been  eluci- 
dated, as  we  have  seen,  through  morphologic  investigation  of  cellular 
activities  taking  place  in  the  animal  body  or  under  controlled  condi- 
tions in  the  test  tube,  and  by  visible  reactions  taking  place  in  test  tubes 
between  the  fluids  of  normal  or  immunized  animals  and  the  bacteria 
and  their  products,  and,  finally,  by  the  more  purely  physiologic  tests 
of  the  protecting  power  and  mechanism  of  action  of  animal  fluids  or 
extracts  when  introduced  into  another  animal  of  the  same  or  different 
species,  along  with  the  bacteria  or  their  products. 

Such  studies  have,  as  is  well  known,  afforded  a  vast  amount  of  in- 
formation. Through  them  the  soluble  secreted  bacterial  poisons  have 
been  demonstrated  and  have  been  found  to  stimulate  the  production  of 
neutralizing  bodies,  the  antitoxins;  bacteria  and  their  culture  filtrates 
have  been  shown  to  call  forth  bodies  which  are  present  in  the  serum  of 
animals  treated  with  them,  and  which  cause  a  precipitation  of  certain 
bacterial  constituents  of  the  filtrate — the  precipitins;  similar  injections, 
have  been  found  to  cause  the  production  of  serum  bodies  which  have 
the  power  of  agglutinating  the  bacteria  when  brought  into  contact  with 
them — the  agglutinins;  and  other  bodies  are  likewise  produced  which 
are  capable  under  proper  conditions  of  killing  the  bacteria — the  bac- 
tericidal substances — or  even  of  dissolving  them  as  we  have  seen  in 
some  instances — the  bacteriolytic  substances.  All  of  these  activities, 
present  to  a  certain  extent  in  normal  serum  but  vastly  increased  in 
immune  sera  may  perhaps,  as  formerly  claimed,  be  due  to  separate 
serum  constituents,  but  it  is  our  own  view  at  present  that  they  repre- 
sent different  activities  of  the  same  specific  type  of  antibody,  namely, 
a  sensitizer  in  the  sense  of  Bordet,  by  the  agency  of  which  the  antigen 
is  rendered  susceptible  either  to  the  action  of  the  alexin  or  to 
phagocytosis.  Each  antigen  injected  into  the  animal  body  would 
thus  give  rise  to  a  specific  sensitizer,  or,  in  the  language  of 
Ehrlich,  amboceptor.  The  union  of  the  antigen  with  this  am- 
boceptor alters  it  so  that,  if  a  whole  cell,  it  is  subjected  to  ag- 
glutination by  electrolytes  and  other  influences;  if  a  dissolved 
protein,  it  becomes  precipitable  and  at  the  same  time  is  rendered 
amenable  to  the   action   of   alexin   and   can  be  more   easily  taken 


FACTS  AND  PROBLEMS  OF  IMMUNITY  311 

Up  by  phagocytic  cells.  The  complement  is  not  increased  during 
immunization. 

These  facts  we  have  learned  from  the  study  of  the  serum;  on  the 
other  hand,  the  morphologic  investigations  instigated  and  carried  on 
largely  by  Metchnikoff  and  his  followers  have  taught  us  the  great  part 
which  the  formed  elements  of  the  blood  and  lymph  play  in  the  protec- 
tion against  and  cure  of  germ  diseases,  and  the  importance  of  the  phago- 
cytes is  now  widely  recognized. 

Of  these  cells,  the  polymorphonuclear  leucocytes  take  a  very  active 
part  in  the  ingestion  and  destruction  of  bacteria,  while  the  large  mono- 
nuclear leucocytes  and  endothelial  cells,  especially  those  lining  the  blood 
vessels  and  body  cavities,  although  also  able  to  ingest  bacteria  directly, 
are  chiefly  active  in  taking  up  cells  of  animal  origin,  principally  those 
which  necessarily,  in  the  normal  course  of  events,  belong  to  the  same 
animal  and  have  probably  become  injured  or  have  suffered  death.  It 
has  also  been  shown  that  many  cells  in  different  parts  of  the  body  may 
take  part  in  phagocytosis  both  under  conditions  of  normal  physiological 
processes  and  under  the  stress  of  invasion.  The  recent  work  of  Kyes^ 
has  inclined  us  to  think  that  phagocytosis  by  fixed  tissue  cells  plays  a 
more  important  part  in  protection  than  hitherto  suspected*.  Kyes  has 
found  that  pigeons  which  possess  a  powerful  normal  resistance  against 
pneumococci,  dispose  of  these  bacteria  by  phagocytosis  carried  on  in 
the  liver  by  endothelial  and  other  cells.  Injected  either  intraperitoneally 
or  intravenously,  the  bacteria  are  soon  found  collected  in  large  masses 
within  such  cells. 

It  does  not  seem,  in  this  connection,  a  far-fetched  idea  to  suppose 
that  phagocytic  cells  may  use  naturally  other  cells  and  bacteria  as  a 
part  of  their  regular  food  supply.  The  polymorphonuclear  leucocytes 
may  thus  depend  to  some  extent  on  the  ever-entering  bacteria  and  their 
remains;  for,  as  we  know,  bacteria  are  constantly  entering  along  the 
regular  channels  of  absorption,  and  it  is  just  as  obvious  that  nimibers 
of  blood  and  tissue  cells  are  constantly  dying  out  and  must  be  disposed 
of,  for  such  processes  are  always  in  evidence  in  the  spleen,  and  the  inges- 
tion of  polymorphonuclear  leucocytes  by  the  large  mononuclears  can  be 
observed  wherever  leucocytes  are  collected  in  exudates,  due  either  to 
infections,  poisons,  or  supposedly  benign  irritants.  The  simple  fact 
that  these  cells  retain  the  basic  physiologic  activities  and  an  ability  to 
ingest  and  digest  food   in   its   crudest  form,  which  ability  was  the 

^Kyes,  Jour,  of  Infect.  Dis.,  March,  1916. 


312  INFECTION  AND  IMMUNITY 

heritage  of  their  free-swimming  ancestors,  and  that  they  have  not 
suffered  the  total  speciaUzation  and  physiologic  degeneracy  of  the  fixed 
tissue  cells,  seems  sufficient  evidence  to  warrant  the  conclusion  that 
they  are  most  active  factors  in  the  protection  of  the  specialized  internal 
tissue  cells,  which  control  the  general  metabolism  and  higher  func- 
tions of  the  animal  body.  It  seems  worth  mentioning  that  the 
leucocytes,  alone  probably  of  all  the  true  cells  of  the  body,  are  entirely 
jndependent  of  the  nerve  control,  and  are  subject  only  to  the  stimula- 
tion of  their  chemical  and  physical  environment,  and  are  thus  sus- 
ceptible of  adaptation  to  and  capable  of  subserving  various  purposes 
which  would  be  fatal  to  the  duties  of  cells  controlled  by  the  nerve 
mechanism  for  the  special  functions  of  the  organism  at  large.  Further 
than  this  the  death  of  leucocytes  does  not  matter,  as  would  the  death  of 
specialized  and  nerve-controlled  cells,  for  no  special  metabolic  or  func- 
tional derangement  occurs  from  their  destruction. 

In  considering  this  independence  of  the  leucocytes  it  must  not  be 
assumed  that  they  have  not  varied  from  primitive  ameboid  cells,  for  un- 
doubtedly their  life  and  proper  functioning  are  largely  determined  by  the 
special  plasma  in  which  they  live,  and  it  may  be  that  their  food,  although 
at  times  crude  compared  with  that  of  the  other  body  cells,  is  never- 
theless usually  prepared  for  them  by  processes  going  on  in  the  plasma. 

Questions  relating  to  the  independence  and  to  the  interrelation  of 
the  plasma  and  leucocytes  in  their  action  on  invading  microorganisms 
and  the  action  of  plasma  as  compared  with  serum  have  been  ground  for 
scientific  strife  for  many  years,  one  side  contending  for  the  activity  of 
the  plasma,  the  other  for  the  activity  of  the  phagocytes;  the  humoralist 
at  first  neglecting,  if  not  absolutely  forgetting,  that"  a  fluid  can  not  be 
self-replenishing,  while  the  supporters  of  phagocytosis  largely  over- 
looked the  fact  that  plasma  is  not  necessarily  an  inert  menstruum  such 
as  salt  solution. 

While  these  differences  have  been  to  some  extent  adjusted  by  the 
theory  and  work  of  Ehrlich,  an  immediate  point  of  contention  is  still  the 
question  of  the  similarity  of  action  of  plasma  and  serum.  The  humoral 
school  contends  that  the  alexin  of  Buchner — complement  of  later 
writers — is  secreted  into  the  plasma,  while  the  Metchnikoff  school 
claims  that  it  is  only  given  up  from  injured  leucocytes  in  the  body, 
and  to  the  serum  by  destruction  of  leucocytes  during  coagulation.  The 
Metchnikoff  school  maintains,  however,  that  the  amboceptors  necessary 
for  bactericidal  and  bacteriolytic  action  are  formed  in  excess  in  the 
phagocytes,  and  given  off  from  these  to  the  plasma,  yet  asserts  that 


FACTS  AND  PROBLEMS  OF  IMMUNITl^  313 

they  are  inactive  for  lack  of  the  complement  which  is  normally  retained 
in  the  leucocytes,  and  that  they  simply  prepare  the  bacteria  for  com- 
plete digestion  in  the  leucocytes.  From  the  work  of  recent  years  it 
would  seem  rather  unlikely  that  either  alexin  or  sensitizer  take  their 
origin  in  the  leucocytes  in  the  cells. 

In  1894  a  further  adjustment  of  differences  took  place,  when  certain 
phenomena  observed  by  Denys  and  his  pupil  Leclef  demonstrated  that 
the  act  of  phagocytosis  when  performed  in  serum,  in  some  instances 
at  least,  was  dependent  on  the  presence  of  certain  substances  in  the 
serum.  Thus,  they  were  able  to  show  that  leucocytes  removed  from 
normal  blood  and  placed  with  bacteria  in  immune  serum  enulfed 
the  bacteria  actively,  while  leucocytes  from  immunized  animals  mixed 
with  bacteria  in  normal  serum  took  up  the  organisms  no  more  actively 
than  the  normal  leucocytes.  The  bodies  inciting  the  phagocytosis  must 
obviously,  then,  they  concluded,  be  in  the  serum.  Whether  these  bodies 
acted  on  the  leucocyte  or  on  the  bacteria  was  not  then  determined, 
but  Denys  concluded,  in  1898,  that  the  bacteria  were  directly  affected. 
The  fact  that  the  action  is  exerted  on  the  bacteria  was  recently  de- 
termined positively  by  Wright  for  normal  serum,  and  by  Neufeld  and 
Rimpau,  independently  of  Wright,  for  immune  serum.  These  bodies 
have  been  called  opsonins  by  Wright,  and  bacteriotropins  by  Neufeld, 
and  have  been  shown  to  attach  themselves  to  the  bacteria  and  thus 
prepare  them  for  ingestion  by  the  phagocytes.  It  has  also  been  shown 
by  various  observers  that  the  more  virulent  the  germ,  the  less  susceptible 
it  is  to  phagocytosis  and  the  more  potent  the  antisera  must  be  to  permit 
of  the  ingestion  by  the  cells. 

If  now,  for  clarity  of  conception,  we  summarize  briefly  the  disease- 
producing  agents  possessed  by  the  bacteria  and  the  opposing  substances 
of  the  serum  and  processes  of  the  animal  body,  we  find  the  true  toxins, 
including  probably  leucocidins  and  hemolysins,  opposed  by  antitoxins 
which  become  free  in  the  plasma;  the  bacterial  bodies  and  possibly  the 
endotoxins  opposed  by  leucocytes,  and  by  lytic  substances  formed  of 
amboceptor  and  complement,  which  either  kill  or  dissolve  the  bacteria 
and  free  the  endotoxins,  but  do  not  neutralize  them;  and,  third,  we 
have  probably  certain  substances  which  oppose  phagocytosis. 

These  last  named  substances  are  largely  problematical.  Bail  has 
described  substances  which  he  calls  aggressins,  which  he  thinks  prevent 
the  destruction  of  bacteria  by  protective  mechanism  of  the  invaded 
body.  He  believes  that  these  are  secretions  produced  by  the  bacteria 
under  the  conditions  found  in  the  normal  body  but  not  in  the  teux, 
21  ^ 


314  INFECTION  AND  IMMUNITY 

tube.  Wassermann  and  Citron  believe  that  Bail's  aggressins  are  noth- 
ing further  than  endotoxins  which  divert  aatibodies  from  bacteria 
themselves,  by  neutralizing  them.  We  ourselves,  reasoning  from  work 
done  by  the  writer  with  Dwyer,  are  inclined  to  believe  that  Bail's 
aggressins  are  of  the  nature  of  proteotoxins  or  anaphylatoxins  which  are 
formed  in  the  reaction  between  bacteria  and  active  serum  constituents 
when  phagocytosis  for  some  reason  is  inhibited.  Whether  or  not  the 
agglutinating  and  precipitating  functions  of  immune  sera  have  any 
protective  importance  is  somewhat  obscure. 

The  recent  work  of  Bull  seems  to  indicate  that  bacteria  are  rapidly 
agglutinating  in  the  animal  body  and  that  by  this  agglutination  phago- 
cytosis of  the  bacteria,  by  fixed  and  mobile  phagocytosis  throughout 
the  body,  is  facilitated. 

While  all  of  these  different  functions  and  chemical  substances  are 
possessed  by  animals  as  a  class,  it  is  becoming  more  and  more  obvious 
that  these  are  not  always  present  or  active  in  the  same  degree,  and  that 
there  are  recognizable  differences  in  the  protective  mechanisms  of  dif- 
ferent animal  species — in  species,  in  fact,  not  far  removed  from  each 
other  in  the  natural  classification.  An  explanation  of  reactions  to  a 
given  infection  which  applies  in  the  case  of  one  species  is  not,  therefore, 
obviously  applicable  in  the  case  of  another  species.  This  is  true  not  only 
of  the  mechanism  of  protection  as  it  takes  place  in  the  serum  of  dif- 
ferent animals  and  in  their  plasma,  but  also  of  phagocytosis  and  phagocy- 
tic digestion  and  the  factors  which  contribute  to  the  perfection  of  these 
processes.  The  constant  stumbling-block  in  the  way  of  a  correct  in- 
terpretation of  processes  going  on  in  the  animal  body  is  our  inability,  as 
we  have  seen,  to  argue  from  serum  phenomena  to  phenomena  occurring 
in  the  plasma.  A  failure  to  keep  this  in  mind,  although  it  is  fully  recog- 
nized, has  undoubtedly  led  to  many  hasty  conclusions,  particularly 
connected  with  the  theory  of  lytic  immunity.  This  may  be  illustrated 
by  a  well-known  example :  Fresh  rabbit  serum  is  actively  germicidal  for 
anthrax  bacilli,  dog  serum  is  not;  yet  rabbits  are  extremely  sensitive 
to  a  true  anthrax  infection,  while  dogs  are  very  resistant.  Experiment 
has  shown  that  there  are  sensitizers  in  the  sera  of  both  these  animals, 
but  that  the  dog's  serum  does  not  contain  the  complement  necessary 
for  their  action  on  the  bacilli;  the  complement  presumably  has  re- 
mained in  the  body  cells,  whereas  in  the  case  of  the  rabbit  it  has  pos- 
sibly been  liberated  from  the  leucocytes  during  clotting.  The  reason 
the  dog  is  insusceptible  is,  then,  not  because  of  plasma  destruction  of 
the  invading  anthrax  germs,  but  probably  because  of  a  more  perfect 


FACTS  AND  PROBLEMS  OF  IMMUNITY  315 

adjustment  of  the  cellular  mechanism  to  the  infection,  although  if  we 
simply  followed  the  theory  of  the  bactericidal  action  of  serum  and 
plasma  as  being  coextensive,  and  the  active  protective  mechanism,  the 
rabbit  should  have  been  protected,  while  the  dog  should  have  suc- 
cumbed. The  difference  here  probably  depends  upon  adequate  phago- 
cytosis in  the  dog,  while  in  the  rabbit  either  the  mechanism  of  ingestion 
is  incomplete  or  the  cells  fail  to  cope  successfully  with  their  contents. 
As  a  matter  of  fact,  we  are  coming  more  and  more  to  the  conclusion 
that  actual  bacteriolytic  processes  within  the  circulating  plasma  are 
much  less  important  in  protection  against  invading  bacteria  than  was 
formerly  supposed  by  the  humoral  school  of  bacteriologists. 

Even  if  the  evidence  so  far  in  our  possession  warranted  the  conclu- 
sion that  the  bactericidal  and  bacteriolytic  bodies  which  are  present 
in  the  sera  of  various  animals  are  present  and  active  against  certain 
microorganisms  in  the  same  manner  in  their  plasma,  we  should,  never- 
theless, still  have  a  number  of  microorganisms  which  are  singularly 
insusceptible  to  such  action  of  the  serum  or  plasma,  even  of  animals 
highly  immunized  against  them.  The  method  of  resistance  against 
these  would  have  to  be  explained  by  a  different  mechanism,  and  if  this 
death  and  destruction  are  not  accomplished  in  the  plasma,  then  we  must 
look  largely  to  the  activities  of  the  leucocytes  for  their  accomplishment. 

Now,  not  only  the  serum  substances  which  further  leucocytosis  have, 
as  we  have  seen,  received  much  attention  of  late,  but  the  bodies  an- 
tagonistic to  the  bacteria  which  are  supposed  to  be  contained  in  the  leu- 
cocytes have  also  been  extensively  investigated. 

Experiments  bearing  on  these  questions  make  it  appear  extremely 
probable  that  bactericidal  and  bacteriolytic  actions  depend  on  two 
processes;  one  of  these  is  the  bacteriolytic  action  of  the  serum  and 
plasma,  the  other  the  bactericidal  action  of  substances  retained  in  the 
leucocytes.  As  an  example  of  the  type  supposed  to  depend  solely  on 
the  bactericidal  substances  of  the  serum  or  plasma,  the  mechanism  of 
the  natural  and  artificial  immunity  of  guinea-pigs  to  typhoid  and  cholera 
may  be  cited,  since  in  these  animals  no  one  has  as  yet  succeeded  in 
demonstrating  that  substances  derived  from  the  leucocytes  by  extrac- 
tion have  any  bactericidal  action  on  the  organisms  of  these  two  diseases. 
This  does  not  mean,  however,  that  the  bactericidal  action  takes  place 
naturally  outside  of  the  leucocytes,  for  the  bacteria  loaded  with  sensi- 
tizers are  probably  taken  into  the  leucocyte  and  there  digested.  As 
examples  of  immunity  depending  on  substances  within  the  leucocytes, 
the  natural  and  artificial  immunity  of  dogs  and  cats  to  anthrax,  and  the 


316  INFECTION  AND  IMMUNITY 

immunity  of  guinea-pigs  to  certain  strains  of  proteus,  may  be  cited, 
for  in  these  cases  the  leucocyte  extract  is  germicidal,  while  the  serum 
is  not. 

Stated  impartially,  then,  our  knowledge  of  immune  bodies  and  proc- 
esses stands  somewhat  thus:  There  are  certain  sensitizing  substances 
possibly  present  in  the  plasma,  certainly  in  the  serum  wherever  this 
is  formed  in  a  pathological  process,  which  have  specific  affinity  for  the 
invading  germs  and  their  products  and  which  when  supplied  with 
complement,  either  normally  present  in  the  plasma  or  derived  from  the 
cellular  source,  may  become  actively  harmful  to  the  bacteria,  killing 
them  or  even  breaking  them  up,  with  the  liberation  of  bodies  which 
may  become  poisonous  to  the  animal  per  se  or  by  further  digestion. 
This  same  sensitization  by  the  plasma  constituents,  while  not  in  itself 
injuring  the  bacteria,  may  subject  them  to  the  phagocytic  power  of 
the  leucocytes.  These  latter  functions  of  the  plasma  are  spoken  of 
as  opsonins  or  hacteriotropins. 

By  a  great  many  workers  it  is  assumed  that  the  opsonic  function  is 
carried  on  by  serum  constituents  entirely  separate  and  apart  from  the 
bactericidal  or  agglutinating  functions.  The  writer  is  himself  inclined 
to  look  upon  the  specific  sensitizing  body  in  the  serum  as  probably 
identical  for  all  these  functions,  in  so  far  as  it  concerns  one  and  the 
same  antibody.  The  antigen  is  sensitized  by  the  specific  antibody  and  is 
then  rendered  subject  to  the  agglutination  or  precipitation,  when 
physical  conditions  are  suitable,  to  bactericidal  effects  in  the  presence 
of  alexin  or  to  phagocytosis  in  the  presence  of  leucocytes. 

After  phagocytosis  has  taken  place,  the  germs  may  be  killed  and 
digested.  Some  of  the  bactericidal  bodies  of  the  phagocytes  are  sub- 
stances differing  in  character  from  the  lytic  bodies  of  the  serum,  and 
are  either  not  given  off  to  the  serum  or  are  not  active  in  it;  whether 
the  lytic  amboceptors  present  in  the  serum  are  derived  from  the  leuco- 
cytes is  doubtful  and  rather  improbable.  This  is  supported  by  the  sup- 
position that  guinea-pig  leucocyte  extracts  are  not  germicidal  for 
cholera  and  typhoid  organisms.  Nevertheless,  intracellular  digestion 
of  these  germs  does  go  on;  it  is  possible,  therefore,  that  the  ambo- 
ceptors present  in  the  plasma,  whatever  their  source,  attach  themselves 
to  the  germs  and  aid  in  intracellular  digestion. 

None  of  the  processes  just  mentioned  leads  to  the  formation  of  anti- 
toxins which  become  free  in  the  plasma  or  serum.  Now,  in  view  of  these 
facts  and  suppositions,  it  may  possibly  be  logical  to  conceive  that  nearly 
all  pathogenic  germs  secrete  bodies  which  are  not  readily  soluble  in  cul- 


FACTS  AND  PROBLEMS  OF  IMMUNITY  317 

ture  fluids  or  in  the  fluids  of  the  animal  body;  that  these  bodies  are  not 
readily,  if  at  all,  assimilable  by  non-phagocyting  cells.  These  bodies 
may,  however,  be  broken  up  by  digestive  bodies  present  in  the  serum, 
and  from  them  may  thus  be  liberated  a  poisonous  substance,  which  may 
then  be  assimilated  by  the  higher  cells  of  the  body,  and,  when  in  suffi- 
cient quantity,  cause  death.  The  more  rapid  the  process  of  liberation 
the  more  quickly  death  ensues.  The  plasma  digestion  is,  then,  according 
to  this  conception,  a  mechanism  which  is  faulty  when  applied  to  bacteria 
and  their  products,  and  if  this  conception  is  correct  the  fault  may  occur 
somewhat  as  follows:  Bacteria  and  their  insoluble  or  non-assimilable 
products  when  taken  into  the  phagocyte  are  subjected  to  two  processes, 
a  primary  bactericidal  and  coagulating  one,  and  then  a  more  leisurely 
lytic  or  disintegrating  action,  during  which  poisonous  products  are 
probably  liberated,  but  slowly  enough  to  be  taken  care  of  by  destroy- 
ing or  neutralizing  bodies.  Even  if  the  leucocyte  dies,  it  is  usually  taken 
up  by  a  mononuclear  cell,  and  the  poisons  do  not  become  free  in  the 
fluids. 

These  are  some  of  the  problems  of  immunity,  particularly  those  re- 
lating to  the  microorganisms  which  are  harmful  to  the  animal  body, 
not  so  much  through  their  ability  to  secrete  harmful  soluble  poisons,  as 
through  their  insistently  invasive  character,  or  by  the  liberation  of  the 
toxic  products  resulting  from  the  destruction  of  their  secretions  or  of 
their  own  bodies.  It  is  the  diseases  caused  by  these  organisms  on  which 
the  attention  of  bacteriologists  is  now  chiefly  centered. 

The  organisms  of  these  diseases  undoubtedly  belong  to  two  or  more 
classes,  in  one  of  whicli  may  be  placed  the  typical  septicemia  producers 
— anthrax,  pneimaococcus,  streptococcus,  etc. — in  the  other  the  less  in- 
vasive organisms,  typified  by  cholera  and  to  some  degree  by  typhoid. 
Between  these  two  extremes  there  are  all  grades. 

If  the  data  amassed  in  the  study  of  these  types  of  microorganisms, 
and  of  the  processes  supposed  to  be  involved  in  meeting  infection  and 
establishing  cure  and  immunity  from  them,  have  been  mafle  clear,  it 
may  be  easier  to  comprehend  some  of  the  problems  which  daily  face 
investigators  in  their  struggle  to  arrive  at  a  rational  method  of  biologic 
treatment,  and  to  realize  more  fully,  in  the  light  of  this  knowledge,  why 
disappointment  has  so  persistently  followed  in  the  wake  of  serum  therapy 
as  applied  to  these  infectious  diseases.  For,  in  spite  of  the  most  persist- 
ent attempts  to  produce  curative  sera,  the  results  have  not  been  satis- 
factory and  have  not  led,  except  in  rare  instances,  to  the  practical  use  of 
such  sera  in  the  treatment  of  disease  in  man. 


31$  INFECTION  AND  IMMUNITY 

The  sera,  thus  produced,  have  not,  except  in  a  very  minor  way, 
been  antitoxic  in  the  usually  accepted  sense,  and  depend,  as  we  have 
seen,  probably,  for  any  protective  value  they  may  possess  slightly  on 
their  germicidal  and  bacteriolytic  power  and  largely  on  the  opsonins 
they  may  carry,  and  thus  facilitate  phagocytosis.  These  sera  are  ca- 
pable of  protecting  an  animal  from  an  infecting  organism,  when  mixed 
with  it  in  surprisingly  minute  quantities;  but  consistent  curative  effects, 
other  than  merely  local,  has  been  definitely  determined  in  a  very  few 
instances  only. 

On  the  other  hand,  indeed,  test  and  experiment  have  shown  that 
animals  and  man  suffering  from  a  true  infection  may  and  often  do 
themselves  furnish  sera  capable  of  strong  bactericidal  and  bacteriolytic 
action  (when  combined  with  normal  sera  containing  complement),  and 
yet  in  spite  of  this,  they  succumb  or  may  be  subject  to  severe  relapses. 

In  the  light  of  these  and  other  facts  which  have  been  cited,  it  seems 
that  one  might  well  refrain  from  attempts  to  produce  beneficial  effects  by 
injecting  still  further  amounts  of  bacteriolytic  or  similar  bodies,  and  seek 
further  for  an  explanation  of  the  exact  methods  and  processes  of  the 
cure  effected  in  those  animals  and  man  who  do  survive  an  infection. 

Failure  to  solve  these  problems  on  lines  hithereo  followed  should  not 
discourage  us,  however,  while  we  know  that  animals  and  man  do  re- 
cover naturally  from  such  infections.  The  conclusion  that  this  power 
must  reside  in  increased  digestive  and  neutralizing  or  poison-destroying 
powers  of  the  animal  organisms  can  not  well  be  avoided,  and  these 
functions  of  the  animal  mechanism  will  probably  be  found  to  take  place 
largely  in  the  wandering  or  fixed  cells. 

The  animal  body,  then,  ideally  protected  in  the  time  of  bacterial  in- 
vasion, may  well  be  one  in  which  some  set  of  cells — phagocytes — are 
immediately  ready  and  able  to  take  up  the  bacterial  invaders  and  de- 
stroy them,  and  within  their  own  bodies  to  neutralize  any  poisons  se- 
creted by  such  invaders  or  arising  from  their  destruction  by  digestion, 
and  this  without  serious  harm  to  the  ingesting  cells;  or — failing  this 
full  immunity  from  serious  harm — it  may  be  that  these  ingesting  cells 
are,  in  their  turn,  taken  up  and,  with  their  noxious  contents,  digested  by 
other  scavenging  cells,  with  a  minimum  liberation  of  the  substances 
which  could  injure  the  body  cells  dedicated  to  specialized  functions. 
The  whole  struggle  of  the  infected  organism  may  be  summed  up  as  a  con- 
flict between  the  leucocytes  and  the  germs,  and  that  it  is  an  attempt  to 
bring  the  invading  germs  within  the  leucocytes,  and  is  a  process  with 
which  the  system  at  large  often  has  little  or  nothing  to  do,  except  as  an 


FACTS  AND  PROBLEMS  OF  IMMUNITY  319 

innocent  and  injured  bystander,  and  that  extracellular  destruction  of 
bacteria  and  toxicogenic  bodies  is  an  untoward  event  after  the  thorough 
establishment  of  infection  often  leading  to  dire  consequences,  and 
depending  on  the  chance  occurrence  of  suitable  digestive  bodies  in  the 
serum  which  have  been  thrown  off  in  excess  from  the  cells,  and  which 
may  thus  become  a  menace  to  the  system  at  large  by  liberating  poison- 
ous bodies  from  comparatively  harmless  compounds. 

Thus,  in  many  instances,  it  seems  we  are  probably  dealing  with  an 
immunity,  a  large  part  of  the  mechanism  of  which  is  intracellular,  not 
only  in  the  sense  of  phagocytosis  and  digestion,  but  in  the  neutralization 
or  destruction  of  poisons  which  arise  from  the  disintegration  of  the  bac- 
teria and  their  products — a  mechanism  in  which  the  protecting  cells 
must  intervene  and,  largely  unaided  by  antitoxic  bodies  in  the  plasma, 
neutralize  or  destroy  within  themselves  the  poisonous  products  of  the  in- 
vading microorganisms. 

It  was  this  thought  which  suggested  the  idea  of  treating  infections 
with  the  extract  of  leucocytes,  and  thus  aiding  the  phagocytes  by 
furnishing  them  as  directly  as  possible  with  the  weapons  which  they 
use  in  their  fight  with  invading  microorganisms,  and  also  to  protect 
them  and  the  cells  of  specialized  function  from  destruction  and  give 
them  an  opportunity  to  recuperate  and  carry  on  successfully  their 
struggle  against  the  invading  germs. 

The  treatment  of  infections  with  vaccines  also  is  based  upon  the 
recognition  of  the  necessity  of  the  direct  intervention  of  phagocytes 
in  the  cure  of  certain  bacterial  diseases  and  is,  as  we  have  seen,  an 
endeavor  to  stimulate  the  production  of  substances  facilitating  the 
ingestion  of  the  organisms  by  the  phagocytes. 

Finally,  it  must  be  remembered  that  while  animal  experiments  are 
necessary,  and  often  extremely  instructive,  one  can,  nevertheless,  not 
always  argue  directly  from  these  to  occurrences  in  man.  An  injection 
disease  is  not  an  infectious  disease,  and  we  are  dealing  usually  with  con- 
ditions in  man  which  are  at  least  not  entirely  analogous  to  artificial  in- 
fections in  animals.  Artificial  infections  are  usually  accomplished  by  an 
abrupt  introduction  of  a  large  quantity  of  infecting  germs  and  their 
products;  the  animal  powers  of  resistance  are  often  immediately  and  se- 
verely taxed;  the  incubation  period  thus  artificially  shortened;  and  the 
germs  themselves,  being  present  in  large  nunbers,  are  not  subjected  to 
such  a  searching  elimination  as  is  usually  the  case  with  the  few  organisms 
gaining  a  foothold  by  the  natural  channels  of  infection.  This  difference 
is  most  marked  in  septicemias,  in  which,  in  animal  experiments,  the  or- 


320  INFECTION  AND  IMMUNITY 

ganisms  have  been  introduced  directly  into  the  circulation  in  quantities 
sufficient  to  bring  about  a  very  rapid  poisoning  and  overwhelming  of  the 
animal,  with  probably  only  a  very  partial  adaptation  of  the  bacteria 
to  the  animal  agents  of  resistance.  On  the  other  hand,  the  septicemic 
invasion  in  man  most  often  follows  the  adaptation  of  the  germs  in  some 
more  favorable  nidus,  and  probably  has  to  do  with  an  evolution  in  the 
bacterial  resistance  to  the  protective  powers,  rather  than  a  decrease  in 
protective  strength  on  the  part  of  man.  Indeed,  both  of  these  processes 
may  increase  hand  in  hand,  and  we  may  have  septicemias  extending  over 
weeks,  months,  and  even  years.  We  may  have,  in  fact,  an  ''armed 
peace''  and  the  prepared  bacterial  army  is  not  to  be  routed  by  the 
application  of  means  which  under  other  circumstances  might  prove  effi- 
cacious, for  we  have  seen  how  the  bacteria  may  possibly  become  resist- 
ant to  the  protective  agents  of  the  animal  body,  and  may  continue  to 
survive  attacks  which  might  well  prove  fatal  to  less  well-adapted 
members  of  their  species.  An  excellent  example  of  this  is  our  own 
recent  experience  with  the  treponema  pallidum  which  in  its  virulent 
condition  resists  serum  influences  which  strongly  agglutinate  the  culti- 
vated non-virulent  strains. 


SECTION  III 

PATHOGENIC  MICROORGANISMS 


CHAPTER  XXI 

THE  STAPHYLOCOCCI   (MICROCOCCI) 

The  power  to  incite  purulent  and  sero-purulent  inflammations  and 
localized  abscesses  in  man  and  animals  is  possessed  by  a  large  variety  of 
pathogenic  bacteria.  Most  infections,  in  fact,  in  which  the  relative 
virulence  of  the  incitant  and  the  resistance  of  the  infected  subject  are 
so  balanced  that  temporary  or  permanent  localization  of  the  infec- 
tious process  takes  place  are  apt  to  be  accompanied  by  the  formation  of 
pus.  The  large  majority  of  acute  and  subacute  purulent  processes, 
however,  are  caused  by  the  members  of  a  well-defined  group  of  bacteria 
spoken  of  as  the  pyogenic  cocci.  Among  these,  pre-eminent  in  import- 
ance, are  the  '' staphylococci"  or  ^' micrococci.^ ^ 

Many  of  the  earlier  investigators  of  surgical  infections  had  seen  small 
round  bodies  in  the  pus  discharged  from  abscesses  and  sinuses  and  had 
given  them  a  variety  of  names.  Careful  bacteriological  studies,  how- 
ever, were  not  made  until  1879  ani  the  years  immediately  following, 
when  Koch,  Pasteur,  Ogston,^  and  others  not  only  described  morphologi- 
cally, but  cultivated  the  cocci  from  surgical  lesions  of  animals  and  man. 
Of  fundamental  importance  are  the  studies  published  by  Rosenbach  ^ 
in  1884,  in  which  the  technical  methods  of  modern  bacteriology  were 
brought  to  bear  upon  this  subject  for  the  first  time.  The  group  of 
staphylococci — so  named  from  their  growth  in  irregular,  grape-like 
clusters^s  made  up  of  several  members,  by  far  the  most  important 

of  which,  pathologically,  is  the  Staphylococcus  pyogenes  aureus. 

~  ■    I  •■•'■■--     • 

'  '   '       ^        »0^s«ow,  Brit.  Med.  Jour.,  1881. 

^Rosenbach,  " Microorganismen  bei  Wundinfektion,"  1884. 
321 


322  PATHOGENIC  MICROORGANISMS 


STAPHYLOCOCCUS  PYOGENES  AUREUS 

Morphology  and  Staining. — This  microorganism,  the  most  frequent 
cause  of  abscesses,  boils,  and  many  surgical  suppurations,  is  a  spherical 
coccus  having  an  average  diameter  of  about  0.8  micra,  but  varying 
within  the  extreme  limits  of  0.4  to  1 .2  micra.  Any  considerable  variation 
from  the  average  size,  however,  is  rare.  The  perfectly  spherical  charac- 
ter may  not  develop,  whenever,  as  is  usually  the  case,  two  or  more  are 


Fia.  69. — Staphylococcus  pyogenes  aureus.    (After  Giinther.) 

grouped  together,  unseparated  after  cell  cleavage.  In  this  case,  adj  acent 
cocci  are  slightly  flattened  along  their  contiguous  surfaces. 

Examined  in  smears  from  cultures  or  pus,  the  staphylococci  may 
appear  as  single  individuals,  in  pairs,  or,  most  frequently,  in  irregular 
grape-like  clusters.  Occasionally,  short  chains  of  three  or  four  may  be 
seen.  In  very  young  cultures  in  fluid  media,  the  diplococcus  form  may 
predominate. 

The  staphylococci  stain  with  all  the  usual  basic  aqueous  anilin  dyes, 
and,  less  intensely,  with  some  of  the  acid  dyes.  Stained  by  the  method 
of  Gram,  they  retain  the  anilin-gentian- violet.  Gram's  method  of 
staining  is  excellently  adapted  for  demonstration  of  these  cocci  in 
tissue  sections. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         323 

Although  exhibiting  marked  Brownian  movements  in  the  hanging 
drop,  staphylococci  are  non-motile  and  possess  no  flagella.  They  are 
non-sporogenous  and  form  no  capsules. 

Cultural  Characters. — Staphylococci  grow  readily  upon  the  usual 
laboratory  media.  The  simpler  media,  made  of  meat  extract,  are  quite 
as  efficient  for  their  cultivation  as  are  the  freshly  made  meat-infusion 


Fig.  70. — Staphylococcus  Colonies. 

products.  The  optimum  temperature  for  staphylococcus  cultivation 
lies  at  or  about  30°  C,  though  growth  readily  takes  place  at  tempera- 
tures as  low  as  15°  C,  and  as  high  as  40°  C.  Slow  but  definite  growth 
has  been  observed  at  a  temperature  as  low  as  10°  C. 

While  development  is  most  characteristic  and  luxuriant  under 
aerobic  conditions,  staphylococci  are  facultatively  anaerobic  on  suitable 
media.    They  grow  readily  in  an  atmosphere  of  hydrogen. 


324  PATHOGENIC  MICROORGANISMS 

As  to  the  reaction  of  media,  staphylococcus  develops  most  favorably 
upon  those  having  a  slightly  alkaline  titer.  Moderately  increased 
alkalinity  or  even  moderate  acidity  of  media  does  not  inhibit  growth. 

On  gelatin  plates,  growth  occurs  readily  at  room  temperature,  form- 
ing within  thirty-six  to  forty-eight  hours,  small,  shining,  pin-head 
shaped  colonies,  appearing,  at  first,  grayish-white,  and  later  assuming  a 
yellowish  hue,  which  intensifies  into  a  light  brown  and  often  a  bronze 
color  as  the  colony  grows  older.  The  intensity  of  the  color  differs  con- 
siderably in  different  races  of  staphylococci.  Liquefaction  of  the  gelatin 
occurs,  and,  shallow,  saucer-shaped  depressions  are  formed  about  the 
colonies  after  forty-eight  hours  or  more.  These  zones  of  fluidification 
grow  larger  as  the  colonies  grow,  finally  becoming  confluent.  Micro- 
scopically, the  colonies  themselves,  before  liquefaction  has  destroyed 
their  outline,  are  round,  rather  finely  granular,  with  smooth  edges. 
They  are  not  flat,  but  rise  from  the  surface  of  the  medium  as  the  seg- 
ment of  a  sphere.  In  gelatin  stab  cultures  in  tubes,  fluidification  leads 
to  the  formation  of  a  funnel-shaped  depression,  with,  finally,  complete 
liquefaction  of  the  medium  and  sedimentation  of  th&  bacteria.  Lique- 
faction of  gelatin  by  the  staphylococcus  is  due  to  a  ferment-hke  body 
elaborated  by  it,  which  is  spoken  of  as  ''gelatinase."  This  substance 
can  be  obtained  apart  from  the  cocci  by  the  filtration  of  cultures.^  It 
is  an  extremely  thermolabile  body. 

On  agar  plates  the  characteristics  of  the  growth,  barring  liquefaction, 
are  much  like  those  on  gelatin.  Colonies  do  not  show  a  tendency  toward 
confluence,  remaining  discrete,  and  show  a  rather  remarkable  differ- 
ence in  the  size  of  the  colonies  occurring  upon  the  same  plate.  Upon 
slanted  agar  in  tubes,  rapid  g|;'owth  occurs,  at  first  grayish-white,  but 
soon  covering  the  surface  of  the  slant  as  a  glistening,  golden-brown 
layer. 

In  broth,  growth  is  rapid,  leading  to  a  general,  even  clouding  of  the 
medium,  and  giving  rise,  after  forty-eight  or  more  hours,  to  the  formation 
of  a  thin  surface  peUicle.  As  growth  increases,  the  bacteria  sink  to  the 
bottom,  forming  a  heavy,  mucoid  sediment.  The  odor  of  old  cultures 
is  often  peculiarly  acrid,  not  unlike  weak  butyric  acid. 

In  milk,  staphylococcus  causes  coagulation  usually  within  three  or 
four  days,  with  the  formation  of  lactic  and  butyric  acids. 

On  potato,  growth  is  abundant,  rather  dry  and  usually  deeply  pig- 
mented. 

>  Loeh,  Cent.  f.  Bakt.,  xxxii,  1902.    • 


STAPHYLOCOCCUS  PYOGENES  AUREUS         325 

Upon  coagulated  animal  sera,  rapid  growth  takes  place  and  eventually 
slight  liquefaction  of  the  medium  is  said  to  occur. 

In  nitrate  solutions,  reduction  of  the  nitrates  to  nitrites  is  caused. 

In  Dunham's  broth,  indol  is  formed. 

In  media  containing  the  carbohydrates — dextrose,  lactose,  or  sac- 
charose— acidification  takes  place  with  the  formation  chiefly  of  lac- 
tic, butyric,  and  formic  acids.  There  is  no  gas  formation,  however. 
In  proteid  media  free  from  sugars,  the  staphylococcus  produces 
alkali. 

The  reducing  action  of  staphylococcus  is  shown  by  decolorization 
in  cultures  of  litmus,  methylene-blue,  and  rosanilin.^ 

Pigment  Formation. — Differentiation  between  the  various  members 
of  the  staphylococcus  group  is  based  largely  upon  the  formation  of 
pigments.  These  pigments,  so  far  as  we  know,  seem  to  be  species 
characteristics.  Thus,  Staphylococcus  pyogenes  aureus  is  recognized 
primarily  by  its  production  of  a  yellowish-brown  pigment,  varying  in 
different  strains  from  a  pale  brown  hue  to  a  deep  golden  yellow.  Pro- 
longed cultivation  upon  artificial  media  may  lead  to  a  diminution  in  the 
depth  of  color  produced.^  It  appears  only  when  cultivation  is  carried 
on  under  freely  aerobic  conditions,  anaerobic  cultivation  resulting  in 
unpigmented  colonies.  The  coloring  matter  is  insoluble  in  water  but 
soluble  in  alcohol,  chloroform,  ether,  and  benzol.^  According  to  Schnei- 
der,* the  pigment  belongs  to  the  class  of  '4ipochromes "  or  fatty  pig- 
ments, and  is  probably  composed  of  carbon,  oxygen,  and  hydrogen, 
without  nitrogen.  Treatment  with  concentrated  sulphuric  acid  changes 
it  to  a  green  or  greenish-blue.^ 

Resistance. — Although  not  spore  formers,  staphylococci  are  more 
resistant  to  heat  than  many  other  purely  vegetative  forms.  The  thermal 
death  point  given  for  Staphylococcus  pyogenes  aureus  by  Sternberg* 
lies  between  56°  and  58°  C,  the  time  of  exposure  being  ten  minutes. 
The  same  author  states  that,  when  in  a  completely  dried  state,  the  coccus 
is  still  more  resistant,  a  temperature  of  from  90°  to  100°  C.  being  re- 
quired for  its  destruction.  Against  low  temperatures,  staphylococci  are 
extremely  resistant,  repeated  freezing  often  failing  to  sterilize  cultures. 

»  Ft.  Muller,  Cent.  f.  Bakt.,  xxvi,  1899. 

2  Fliigge,  "Die  Microorg.,"  etc. 

3  Migula,  "System  d.  Bakt.,"  Jena,  1897. 

*  Schneider,  Arb.  a.  d.  bakt.  Inst.,  Karlsruhe,  1,  vol.  i,  1894. 
« Fischer,  "  Vorles.  fiber  die  Bakt.,"  Jena,  1903. 
^Sternberg,  "Textbook,"  etc.,  N.  Y.,  1901,  p.  375. 


326  PATHOGENIC  MICROORGANISMS 

Desiccation  is  usually  well  borne,  staphylococci  remaining  alive  for 
six  to  fourteen  weeks  when  dried  upon  paper  or  cloth.*  On  slant  agar, 
staphylococci  may  be  safely  left  for  three  or  four  months  without  trans- 
plantation, and  remain  alive.^ 

The  resistance  of  staphylococci  to  chemicals,  a  question  of  great 
surgical  importance,  has  been  made  the  subject  of  extensive  researches, 
notably  by  Liibbert,^  Abbott,^  Franzott,^  and  many  others.  According 
to  Liibbert,  inhibition  of  staphylococcus  growth  is  attained  by  the  use  of 
boric  acid  1  in  327,  salicylic  acid  1  in-  650,  corrosive  sublimate  1  in 
80,000,  carbolic  acid  1  in  800,  thymol  1  in  11,000.  Staphylococci  are 
killed  by  corrosive  sublimate  1  in  1,000  in  ten  minutes,  by  carbolic  acid 
1  per  cent  in  35  minutes,  3  per  cent  in  2  minutes  (Franzott).  Ethyl 
alcohol,®  even  when  absolute,  is  not  very  efficient  as  a  disinfectant. 
Nascent  iodin,  as  split  off  from  iodoform  in  wounds,  is  extremely  power- 
ful in  destroying  staphylococci. 

Pathogenicity. — Separate  strains  of  Staphylococcus  pyogenes  aureus 
show  wide  variations  in  relative  virulence.  The  most  highly  virulent 
are  usually  those  recently  isolated  from  human  suppurative  lesions, 
but  no  definite  rule  can  be  formulated  in  this  respect.  The  virulence 
of  a  given  strain,  furthermore,  may  be  occasionally  enhanced  by  re- 
peated passages  through  the  body  of  a  susceptible  animal.  Prolonged 
cultivation  upon  artificial  media  is  liable  to  decrease  the  virulence  of  any 
given  strain,  though  this  is  not  regularly  the  case.  There  are,  more- 
over, unquestionably,  many  staphylococci  constantly  present  in  the  air, 
dust,  and  water,  which  although  morphologically  and  culturally  not 
unlike  the  pathogenically  important  species,  may  be  regarded  as 
harmless  saprophytes. 

The  susceptibility  of  animals  to  staphylococcus  infection  is, 
likewise,  subject  to  extreme  variations,  depending"  both  upon  differ- 
ences between  species  and  upon  fortuitous  individual  differences 
in  susceptibility  among  animals  within  the  same  species.  Animals 
on  the  whole  are  less  susceptible  to  staphylococcus  than  is  man. 
Among  the  ordinary  laboratory  animals,  rabbits  are  most  sus- 
ceptible   to    this   microorganism.      Mice,    and    especially    the    white 


1  Deslongchamps,  Paris,  1897. 

^Passet,  Fort.  d.  Med.,  2  and  3,  1885. 

» Liibbert,  "Biol.  Untersuch.,"  Wurzburg,  1886. 

*  Abbott,  Medical  News,  Phila.,  1886. 

*  Franzott,  Zeit.  f.  Hyg.,  1893. 
^Hanel,  Beit.  z.  klin.  Chir.,  xxvi 


STAPHYLOCOCCUS  PYOGENES  AUREUS         327 

Japanese  mice,  show  considerable  susceptibility.     Guinea-pigs  possess 
a  relatively  higher  resistance/ 

Subcutaneous  or  intramuscular  inoculation  of  a  susceptible  animal 
usually  results  in  the  formation  of  a  localized  abscess  with  much  pus 
formation  and  eventual  recovery.  Intraperitoneal  inoculation  is  more 
often  fatal.  Intravenous  inoculation  of  doses  of  0.5  c.c,  or  more,  of 
fresh  broth  cultures  of  virulent  staphylococci  usually  leads  to  pyemia 
with  the  production  of  secondary  abscesses,  located  chiefly  in  the  kid- 
neys and  the  heart  and  voluntary  muscles,  but  not  infrequently  in 
other  organs  as  well.  In  the  kidney  they  occur  as  small  foci,  situated 
most  often  in  the  cortex,  composed  of  a  central,  necrotic  pus  cavity, 
surrounded  by  a  zone  of  acute  inflammatory  exudation.  Staphylo- 
coccus lesions  form  histologically  the  typical  "acute  abscess."  Not 
infrequently  the  pyemic  condition  is  accompanied  by  suppurative 
lesions  in  the  joints.  Intravenous  injections  of  virulent  staphylococci 
preceded  by  injury  to  a  bone  is  often  followed  by  the  development  of 
osteomyelitis.  Mechanical  or  chemical  injury  of  the  heart  valves 
preceding  intravascular  staphylococcus  inoculation  may  result  in 
localization  of  the  infection  on  or  about  the  heart  valves,  leading 
to  "malignant  endocarditis."  The  pyemic  conditions  following  staphy- 
lococcus inoculation  usually  lead  to  chronic  emaciation  and  death 
after  an  interval  dependent  upon  the  relative  virulence  of  the  micro- 
organism, the  amount  injected,  and  the  resistance  of  the  infected 
subject.  Large  doseS  of  unusually  virulent  cultures  cause  death  within 
twenty -four  hours,  or  even  less,  without  abscess  formation. 

As  above  stated,  the  susceptibility  of  man  to  spontaneous  staphy- 
lococcus infection  is  decidedly  more  marked  than  is  tliat  of  animals. 
The  form  of  infection  most  frequently  observed  is  the  common  boil 
or  furuncle.  As  Garre,^  Biidinger,^  Schimmelbusch,*  and  others  .  Jiave 
demonstrated  by  experiments  upon  their  own  bodies,  energetic  rubbing, 
of  the  skin  with  virulent  staphylococcus  cultures  may  often  be  followed 
by  the  development  of  a  furuncle.  Subcutaneous  inoculation  of  the, 
human  subject  invariably  gives  rise  to  an  abscess.  The  pathological 
lesions  which  may  be  produced  in  man  by  virulent  staphylococqi  are. 
naturally  of  great  variety,  depending  upon  the  mode  of  inoculation,  and 

1  Terin,  Ref.  in  Lubarsch  und  Ostertag,  Ergebnisse,  1896;  Lingelsheim,  "AetioL 
d.  Staph.  Inf.,"  etc.,  Wien,  1900. 

^Garre,  Beit.  z.  klin.  Chir.,  x,  1893. 

3  Budinger,  Lubarsch  und  Ostertag,  Ergebnisse,  etc.,  1896. 

* Schimmelbusch,  Ref.  by  Budinger. 


328  PATHOGENIC  MICROORGANISMS 

upon  the  relation  between  the  virulence  of  the  incitant  and  the  resist- 
ance of  the  subject.  Apart  from  the  formation  of  localized  abscesses, 
staphylococci  are  common  as  the  incitants  of  surgical  suppurations 
and  wound  infections.  The  large  majority  of  acute  suppurative  in- 
flammations of  bone  (osteomyelitis)  are  caused  by  staphylococci.  Ab- 
scesses of  the  brain,  of  the  liver,  and  of  the  lung  may  be  due  to  this 
microorganism.  It  may  give  rise  to  ascending  infections  of  the  genito- 
urinary tract,  leading  to  pyelonephritis.  Empyema  or  peritonitis  may 
be  caused  by  its  entrance  into  the  serous  cavities  from  the  lung  or 
bowel.  When  gaining  achess  to  the  circulation  from  some  localized 
focus,  it  gives  rise  to  septicemia  and  may  lead  to  malignant  endocarditis 
and,  by  secondary  locaUzation  in  the  viscera,  to  general  pyemia.  As 
the  incitant  of  septicemia  it  can  frequently  be  found  by  blood  culture 
during  the  life  of  the  patient.  Puerperal  sepsis  is  not  infrequently  a 
staphylococcus  disease.  Of  recent  years  several  authors  have  claimed 
direct  etiological  relationship  for  the  Staphylococcus  pyogenes  aureus 
with  acute  articular  rheumatism.^  While  not  unlikely,  this  claim  is 
not,  at  present,  substantiated  by  sufficiently  exact  evidence. 

Apart  from  the  local  inflammatory  reactions  called  forth  by 
staphylococcus  invasion,  all  such  infections,  if  severe  or  prolonged, 
give  rise  to  profound  toxic  manifestations  evidenced  by  characteris- 
tically irregular  temperature  (the  so-called  "septic  type"),  by  head- 
ache, nausea,  and  general  malaise,  and  not  infrequently  by  chills. 
Prolonged  chronic  infection  with  staphylococci  may  give  rise  to  the 
So-called  amyloid  changes  in  liver,  spleen,  and  kidneys. 

Toxic  Products. — Endotoxins. — The  dead  bodies .  of  staphylococci 
injected  into  animals  may  occasionally  give  rise  to  abscess  formation, 
and,^  if  in  sufficient  quantity,  may  cause  death.  To  obtain  the  latter 
result,  however,  large  quantities  are  necessary,  the  endotoxic  substances 
within  the  dead  cell  body  of  these  microorganisms  being  probably  neither 
very  poisonous  nor  abundant.^ 

That  dead  cultures  of  Staphylococcus  aureus  exert  a  strong  positive 
chemotaxis  for  leucocytes  was  shown  beyond  question  by  the  experi- 
ments of  Borissow.* 

Hemolysins. — In    1900    Kraus^  noticed   the   hemolytic    action    of 

^A.H.  Weis,  Inaug.-Diss.,  Berlin,  1901. 

^  Schattenfroh,  Arch.  f.  Hyg.,  xxxi,  1887. 

8  V.  Lingelsheim,  "  Aetiol.  u.  Therap.  d.  Staph.  Krank.,"  Wien,  1900. 

*Bori8sow  Zieglers  Beitr.,  xvi,  1894. 

^Xraus,  Wien.  klin.  Woch,,  iii^  190D. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         329 

staphylococci  growing  upon  blood-agar  plate  cultures.  Neisser  and 
Wechsbergi  then  showed  that  this  hemolytic  substance,  secreted  by  the 
staphylococcus,  could  be  demonstrated  in  filtrates  of  bouillon  cultures. 
Such  hemolysins  are  produced  by  Staphylococcus  aureus,  and,  to  a 
lesser  degree,  by  Staphylococcus  albus.  The  quantity  produced  varies 
enormously  with  different  strains  and  seems  to  be  roughly  proportionate 
to  the  virulence  of  the  particular  microorganism,  though  exceptions  to 
this  rule  are  not  uncommon.  Absolutely  avirulent  races  do  not,  so 
far  as  we  know,  produce  hemolysins.  The  culture  medium  most  favor- 
able to  the  formation  of  these  substances  is,  according  to  Neisser  and 
Wechsberg,  a  moderately  alkaline  beef  bouillon.  Cultivated  at  37.5°  C, 
the  bouillon  contains  the  maximum  amount  of  hemolytic  substance  be- 
tween the  eighth  and  fourteenth  day,  and  this  may  be  separated  from 
the  bacteria  by  filtration  through  Berkefeld  or  Chamberland  filters. 

The  hemolytic  action  may  be  observed  by  the  general  technique  for 
determining  hemolysis  (given  on  page  259) .  It  is  important  to  wash 
the  red  blood  corpuscles  used  for  the  experiments,  since  many  animals 
normally  possess  small  quantities  of  antihemolysin  in  their  blood-sera 
(man  and  horse  especially)  .^  The  red  blood  corpuscles  of  rabbits,  dogs, 
and  guinea-pigs  are  extremely  susceptible  to  the  action  of  the  staphylo- 
hemolysin.  Those  of  man  are  less  easily  injured  by  it.  The  hemolytic 
action  takes  place,  as  Todd^  and  others*  have  shown,  not  only  m 
vitro,  but  in  the  living  animal  as  well. 

The  staphylo-hemolysin  is  comparatively  thermolabile.  According 
to  Neisser  and  Wechsberg,  heating  it  to  56°  C.  for  twenty  minutes  de- 
stroys it.  According  to  some  other  authors,  however,  higher  tempera- 
tures (60°  to  80°  C.)  are  required.  Reactivation  of  a  destroyed  staphylo- 
hemolysin  has  so  far  been  unsuccessful.  The  fact  that  antistaphylolysin 
is  occasionally  present  in  normal  sera  has  been  mentioned  above.  This 
antibody  is  most  abundant  in  the  blood  of  horses  and  of  man.  Arti- 
ficially antistaphylolysin  formation  is  easily  induced  by  subcutaneous 
inoculation  of  staphylolysin  into  rabbits. 

Leucocidin. — In  1894,  Van  de  Velde  ^  discovered  that  the  pleural 

exudate  of  rabbits  following  the  injection  of  virulent  staphylococci 

showed  marked  evidences  of  leucocyte  destruction.  He  was  subse- 
■  — ~ — I         ■   I 

»  Neisser  und  Wechsberg,  Zeit.  f.  Hyg.,  xxxvi,  190L 

^Neisser,  Deut.  med.  Woch.,  1900. 

3  Todd,  Trans.  London  Path.  Soc,  1902. 

^Kraus,  Wien.  klin.  Woch.,  1902. 

6  Van  de  Velde,  La  Cellule,  x,  1894  ^ 

22 


330  PATHOGENIC  MICROORGANISMS 

quently  able  to  show  that  the  substance  causing  the  death  and  partial 
solution  of  the  leucocytes  was  a  soluble  toxin  formed  by  the  staphylo- 
coccus, not  only  in  vivo,  but  in  vitro  as  well;  for  cultures  of  Staphylo- 
coccus pyogenes  aureus,  grown  in  mixtures  of  bouillon  and  blood 
serum,  contained,  within  forty-eight  hours,  marked  quantities  of  this 
'^eucocidin." 

Other  workers  since  Van  de  Velde  have  evolved  various  methods  for 
obtaining  potent  leucocidin.  Bail*  obtained  it  by  growing  virulent 
staphylococcus  in  mixtures  of  one-per-cent  glycerin  solutions  and  rab- 
bit serum.  Neisser  and  Wechsberg^  advise  the  use  of  a  carefully  titrated 
alkaline  bouillon.  To  obtain  the  leucocidin  l^e  from  bacteria,  the 
cultures  are  passed  through  Chamberland  or  Berkefeld  filters,  after 
about  eight  to  eleven  days'  growth  at  37°  C,  at  which  time  the  con- 
tents in  leucocidin  are  usually  at  their  highest  point. 

The  action  of  leucocidin  upon  leucocytes  may  be  observed  in  vivo 
by  the  pimple  method  of  Van  de  Velde,  of  injecting  virulent  staphylo- 
cocci intrapleurally  into  rabbits  and  examining  the  exudate.  Bail 
advises  the  production  of  leucocytic  intrapleural  exudates  by  the  use 
of  aleuronat  and  following  this  after  twenty-four  hours  by  an  injection 
of  leucocidin-filtrate.  In  vitro  the  phenomenon  may  be  observed  by 
direct  examination  of  mixtures  of  leucocytes  and  leucocidin  in  the 
hanging  drop  on  a  warmed  stage,  or  by  the  '^methylene-blue  method'', 
of  Neisser  and  Wechsberg.  This  method  is  based  upon  the  fact  that 
living  leucocytes  will  reduce  methylene-blue  solutions  and  render  them 
colorless,  while  dead  leucocytes  have  lost  this  power.  Leucocidin  and 
leucocytes  are  allowed  to  remain  in  contact  for  a  given  time  and  to  them 
is  then  added  an  extremely  dilute  solution  of  methylene-blue.  If  the 
leucocytes  have  been  actively  attacked  by  leucocidin,  no  reduction  takes 
place.     This  method  is  particularly  adapted  for  quantitative  tests. 

All  staphylococcus  strains  do  not  produce  leucocidin  to  the  same 
degree.  Almost  all  true  Staphylococcus  pyogenes  aureus  cultures 
produce  some  of  this  toxin,  but  one  strain  may  produce  fifty-  and  a 
hundred-fold  the  quantity  produced  by  another.  Staphylococcus 
pyogenes  albus  gives  rise  to  this  substance  but  rarely,  and  then  in  small 
quantity. 

Leucocidin  seems  to  be  similar  to  the  soluble  toxins  of  other  bacteria. 
It  is  rapidly  destroyed  by  heat  at  58°  C,  and  deteriorates  quickly  in 


iBail,  Arch.  f.  Hyg.,  xxxii,  1898. 

^Neisser  und  Wechsberg,  Zeit.  f.  Hyg.,  xxxvi,  1901. 


STAPHYLOCOCCUS  PYOGENES  AUREUS         331 

Culture  fluids  at  incubator  temperatures.  It  is  distinct  from  staphylo- 
hemolysin  as  shown  by  differences  in  thermostability. 

Soon  after  Van  de  Velde's  discovery  of  leucocidin,  Denys  and  Van 
<ie  Velde  ^  produced  an  antileucocidin  by  treating  rabbits  with  pleural 
exudate  containing  leucocidin.  Neisser  and  Wechsberg  ^  later  confirmed 
these  results  and  showed  that  among  staphylococci,  leucocidin  is  not 
specific,  the-  toxin  of  all  strains  of  Staphylococcus  aureus  and  albus 
examined  being  neutralizable  by  the  same  antileucocidin.  Antileuco- 
cidin is  often  found  in  the  normal  sera  of  hocees  and  man.^ 

Leucocidin  should  BB&t  be  confounded  with  "leucotoxin,"  a  substance 
obtained  in  serum  by  treatment  of  animals  with  leucocytes,  a  true 
"cytotoxin,"  having  no  connection  Whatever  with  the  staphylococcus. 

Staphylococci,  besides  the  toxic  substances  already  mentioned,  give 
rise  to  gelatinase^  spoken  of  in  the  section  upon  cultivation,  and  to  a 
proteolytic  ferment  by  means  of  which  albuminous  media  (Loeffler's 
serum)  may  be  slightly  digested. 

Immunization. — Animals  can  be  rendered  actively  immune  by  re- 
peated inoculations  with  carefully  graded  doses  of  living  or  dead  staphy- 
lococcus cultures.^  The  production  of  antistaphylolysin  and  of  anti- 
leucocidin in  the  sera  of  animals  so  treated,  has  been  alluded  to  in  the 
preceding  sections.  The  sera  of  such  actively  immunized  animals 
possess  distinct  protective  power  when  administered  to  other  animals, 
slightly  before  or  at  the  same  time  with  an  inoculation  of  staphylo- 
cocci. •  They  do  not,  however,  exhibit  very  high  bactericidal  power  in 
vitro,  the  protective  properties  depending  probably  entirely  upon  their 
opsonic  contents.^ 

Agglutinins  have  been  demonstrated  in  staphylococcus  immune  sera 
by  a  number  of  authors,  and  have  been  of  some  slight  value  in  differ- 
entiating between  the  several  groups  of  staphylococci.®  A  rather  sur- 
prising result  of  these  researches  has  been  the  recognition  that  immune 
sera  obtained  with  pathogenic  staphylococci  will  agglutinate  other 
pathogenic  staphylococci,  whether  belonging  to  the  group  of  Staphy- 
lococcus pyogenes  aureus  or  that  of  Staphylococcus  pyogenes  albus, 

1  Denys  et  Van  de  Velde,  La  Cellule,  xi,  1895. 

2  Loc.  cit.  : 
'  Van  de  Velde,  Presse  m^dicale,  i,  1900. 

*  Richet  et  Hericourt,  Compt.  rend,  de  Tacad.  des  sci.,  cvii,  1888. 
5  Kolle  und  Otto,  Zeit.  f .  Hyg.,  xli,  1902. 

^  Proscher,  Cent.  f.  Bakt.,  xxxiv,  1903;  v.  Ldngelsheim,  "Aetiol.  u.  Therap.  d. 
Staphyl.,"  etc.,  Wien,  1900. 


332  PATHOGENIC  MICROORGANISMS 

but  will   not    agglutinate  any  of  the    non-pathogenic    members    of 
either  group.  ^ 

Active  immunization  of  human  beings  suffering  from  staphylococcus 
infections  has  been  extensively  practiced  by  Wright,  in  connection  with 
his  work  on  opsonins.  There  can  be  no  question  about  the  fact  that  the 
opsonic  substances  in  the  blood  are  increased  by  the  injection  of  dead 
staphylococci.  The  procedure  is  of  therapeutic  value  in  subacute  and 
chronic  cases.  The  work  of  Hiss  on  the  use  of  leucocyte  extracts  in 
animals  infected  with  Staphylococcus  pyogenes  aureus  has  given  en- 
couragement for  such  treatment  in  human  beings.  A  number  of 
staphylococcus  infections  in  man  have  been  successfully  treated  with 
leucocyte  extract  by  Hiss  and  Zinsser. 

STAPHYLOCOCCUS  PYOGENES  ALBUS 

This  coccus  differs  from  Staphylococcus  pyogenes  aureus  simply  in 
the  absence  of  the  golden  yellow  coloration  of  its  cultures.  Morpho- 
logically, culturally,  and  pathogenically,  it  is  in  every  way  identical 
with  the  staphylococcus  described  in  the  preceding  section,  but  its 
toxin-  and  enzyme-producing  powers  in  general  are  less  developed  than 
those  of  the  aureus  variety.  Its  close  biological  relationship  to  aureus 
is  furthermore  demonstrated  by  its  agglutination  in  Staphylococcus 
pyogenes  aureus  immune  sera. 

STAPHYLOCOCCUS  EPIDERMIDIS  ALBUS 

The  Staphylococcus  epidermidis  albus  described  by  Welch  is  merely 
one  of  the  non-pathogenic  varieties  of  Staphylococcus  pyogenes  albus 
and  possibly  does  not  deserve  separate  classification.  It  may  give  rise 
to  unimportant  stitch  abscesses. 

STAPHYLOCOCCUS  PYOGENES  CITREUS 

Staphylococcus  pyogenes  citreus  produces  a  bright  yellow  or  lemon- 
colored  pigment  of  distinctly  different  hue  from  that  of  Staphylococcus 
pyogenes  aureus.  It  may  be  pyogenic  and  in  every  way  similar  to 
Staphylococcus  pyogenes  aureus,  but  is  less  often  found  in  con- 
nection with  pathological  lesions  than  either  of  the  preceding  staphy- 
lococci. 

1  Proscher,  Deut.  med.  Woch.,  xi,  1903. 


STAPHYLOCOCCUS,   PYOGENES    CITREUS  333 

A  large  number  of  staphylococci,  differing  from  those  described 
above  in  one  or  another  detail,  have  been  observed.  They  are  of  com- 
mon occurrence  and  are  met  with  chiefly  as  contaminations  in  the  course 
of  bacteriological  work.  Few  of  these  have  any  pathological  significance 
and  none  of  them  are  toxin-producers,  so  far  as  we  know.  Many  of  them 
differ,  furthermore,  in  their  inability  to  liquefy  gelatin.  All  of  them 
belong  more  strictly  to  the  field  of  botany  than  to  that  of  patho- 
logical bacteriology. 

Atypical  pathogenic  staphylococci  have  been  described  by  a  number 
of  observers.  Thus  Weichselbaum  ^  isolated  a  staphylococcus  from  a 
case  of  malignant  endocarditis  which  could  not  be  cultivated  at  room 
temperature,  and  grew  only  in  very  delicate  colonies.  Veillon,^  moreover, 
has  described  a  strictly  anaerobic  staphylococcus  cultivated  from  the 
pus  of  an  intra-abdominal  abscess. 


MIGROGOGGUS  TETRAGENUS 

In  1881,  Gaffky  ^  discovered  a  micrococcus  which  occurs  regularly 
in  groups  of  four  or  tetrads.  He  first  isolated  it  from  the  pus  discharged 
by  tuberculous  patients  with  pulmonary  lesions.  Observed  in  smear 
preparations  from  pus,  the  tetrads  are  slightly  larger  in  size  than  the 
ordinary  staphylococcus,  flattened  along  their  adjacent  surfaces,  and 
surrounded  by  a  thick  halo-like  capsule.  Preparations  from  cultures 
often  lack  these  capsules.  The  micrococcus  is  easily  stained  by  the 
usual  basic  anilin  dyes.  Stained  by  Gram's  method,  it  is  not  decolor- 
ized, retaining  the  gentian-violet. 

Gultivation. — Micrococcus  tetragenus  grows  on  the  ordinary  labora- 
tory media,  showing  a  rather  more  delicate  growth  than  do  the  staphy- 
lococci. 

On  agar,  the  colonies  are  at  first  transparent,  later  they  become 
grayish-white,  but  are  always  more  transparent  than  are  staphylococcus 
cultures. 

On  gelatin,  growth  is  rather  slow  and  no  liquefaction  takes  place. 

Broth  is  evenly  clouded. 

On  potato  there  is  a  white,  moist  growth  which  shows  a  tendency  to 
confluence. 

»  Weichselhaum,  Baumgarten  Jahresb.,  1899,  Ref. 

2  Veillon,  Compt.  rend.  soc.  de  biol,,  1893. 

» Gaffky,  Mitteil.  a.  d.  kais.  Gesimdheitsamt,  i,  1881. 


334 


PATHOGENIC  MICROORGANISMS 


Milk  is  coagulated  and  litmus  milk  indicates  acid  formation. 

Pathogenicity. — Micrococcus  tetragenus  is  especially  pathogenic 
for  Japanese  mice,  which  succumb  within  three  or  four  days  to  subcuta- 
neous inoculation.^  Gray  mice,  rats,  guinea-pigs,  and  rabbits  are  less 
susceptible,  showing  only  a  localized  reaction  at  the  point  of  inoculation. 


Fig.  71. — Micrococcus  Tetragenus.     (In  spleen  of  infected  mouse.) 

The  organism  has  occasionally  been  isolated  from  spontaneous  abscesses 
observed  in  domestic  animals. 

In  man,  this  microorganism  is  usually  found  without  any  particular 
pathogenic  significance,  in  sputum  or  saliva.  In  isolated  cases,  how- 
ever, it  has  been  described  as  the  sole  incitant  of  abscesses. 

Bezangon  ^  has  isolated  Micrococcus  tetragenus  from  a  case  of  menin- 
gitis. A  single  case  of  tetragenus  septicemia  is  on  record,  reported  in 
1905  by  Forneaca.3 

In  America,  this  microorganism  has  not  been  frequently  observed  in 
connection  with  disease.  It  is  often  found,  however,  in  considerable 
numbers,  in  smears  of  sputum  which  are  being  examinee!  for  pneumo- 
cocci  or  tubercle  bacilli. 


1  Mailer,  Wien.  klin.  Woch.,  17,  1904. 

2  Bezangon,  Semaine  m6d.,  1898. 
»  Fomeacat  Rif .  med.,  1903. 


CHAPTER  XXII 

THE  STREPTOCOCCI 

Among  the  pyogenic  cocci,  there  is  a  large  and  important  group  of 
organisms  which  multiply  by  division  in  one  plane  of  space  only,  and 
thus  give  rise  to  appearances  not  unlike  chains  or  strings  of  beads. 
The  term  streptococcus  or  chain-coccus  is,  therefore,  a  purely  morpho- 
logical one  which  includes  within  its  limits  microorganisms  which  may 
differ  from  each  other  considerably,  both  as. to  cultural  and  pMhogenic 
properties.  Thus,  cocci  which  form  chains  may  be  isolated  from  water, 
milk,  dust,  and  the  feces  of  animals  and  man.  These  may  have  little 
but  their  morphological  appearance  in  common  with  the  pyogenic 
streptococci  which  are  so  important  as  the  incitants  of  disease.  The 
interrelationship  between  streptococci  from  different  sources,  how- 
ever, is  by  no  means  fully  understood,  and  we  are  forced  at  present  to 
content  ourselves  with  the  recognition  of  a  large  morphological  group, 
in  no  individual  case  taking  the  pathogenic  or  more  special  cultural 
characteristics  for  granted. 

STREPTOCOCCUS   PYOGENES 

Of  paramount  importance  among  the  streptococci  are  those  which 
possess  the  power  of  giving  rise  to  disease  processes  in  animals  and  in, 
man,  and  which,  because  of  their  frequent  association  with  suppura- 
tive inflammations,  are  roughly  grouped  under  the  heading  of  Strep- 
tococcus pyogenes. 

The  same  researches  upon  surgical  infections  which  led  to  the  dis- 
covery of  the  staphylococci,  laid  the  basis  for  our  knowledge  of  the 
streptococci.  The  fundamental  studies  of  Pasteur  and  Koch*  were  fol- 
lowed, in  1881,  by  the  work  of  Ogston,^  who  was  the  first  to 
differentiate  between  the  irregularly  grouped  staphylococci  and  the 
chain-cocci. 

1  Koch,  "  Untersuch.  iiber  Wundinfektion,"  etc.,  1878< 

2  Oyston,  Brit.  Med.  Jour.,  1881. 

335 


336 


PATHOGENIC  ORGANISMS 


Pure  cultures  of  streptococci  were  first  obtained  by  Fehleisen*  in  1883 
and  by  Rosenbach^  in  1884.  The  thorough  and  systematic  researches  of 
the  last-named  authors,  together  with  those  of  Passet,^  were  of  special 


.  ,        * 

-■■>          ■      i 

Av.^ 

'■  1 

^      .-• 

/  ■ 

/     V 

f 

t                  ^ 

./ 

v^^;.^--.  . 

! 

/ 

* 

Fig.  72. — Streptococcus  pyogenes. 

influence  in  placing  our  knowledge  of  the  pathogenic  properties  of 
streptococci  upon  a  scientific  basis. 

Morphology  and  Staining. — ^The  individual  streptococcus  is  a  spherical 
microorganism  measuring  from  0.5  micron  to  1  micron  in  diameter. 
Since  the  line  of  cleavage  of  cocci,  when  in  chains,  is  perpendicular  to  the 


^Fehleisen,  "  Aetiol.  d.  Erysipelas,"  Berlin,  1883. 

"  Rosenbach,  "  Mikroorg.  bei  Wundinfektion,"  etc.,  Wiesbaden,  1884. 

8  Passet,  "  Untersuch.  iiber  die  eitrigen  Phlegm.,"  etc.,  Berlin,  1885. 


STREPTOCOCCUS  PYOGENES  337 

long  axis  of  the  chain,  adjacent  cocci  often  show  slight  flattening  of  the 
contiguous  surfaces,  forming,  as  it  were,  a  series  of  diplococci  arranged 
end  to  end.  As  a  general  rule  the  streptococci  pathogenic  for  man, 
when  grown  upon  favorable  media,  have  a  tendency  to  form  chains 
made  up  of  at  least  eight  or  more  individuals,  while  the  more  saprophy- 
tic,, less  pathogenic  varieties  are  apt  to  be  united  in  shorter  groups. 
Upon  this  basis  a  rough  morphological  distinction  has  been  made  by  v. 
Lingelsheim,^  who  first  employed  the  terms  Streptococcus  "longus" 
and  "brevis."  A  differentiation  of  this  kind  can  hardly  be  re- 
lied upon,  however,  since  the  length  of  chains  is  to  some  degree  de- 
pendent upon  cultural  and  other  environmental  conditions.  Species 
which  exhibit  long  and  tortuous  chains,  when  grown  upon  suitably 
alkaline  bouillon,  or  ascitic  broth,  may  appear  in  short  groups  of  three  or 
four,  or  even  in  the  diplo  form,  when  cultivated  upon  solid  media  or 
unfavorable  fluid  media.  Stained  specimens  often  show  swelling  and 
enlargement  of  individual  cocci,  giving  the  chains  an  irregularly  beaded 
appearance.  These  swollen  individuals  are  probably  to  be  interpreted  as 
involution  forms  and  are  seen  with  especial  frequency  in  old  cultures. 
Streptococci  do  not  form  spores,  are  non-motile,  and  do  not  possess 
flagella.  There  can  be  no  doubt  that  certain  species  of  true  streptococci 
may  possess  capsules,  though  these  are  not  so  regularly  demonstrable 
and  are  more  delicately  dependent  upon  cultural  conditions  than  are  the 
capsules  of  the  pneumococci.^  The  capsulated  streptococci  will  be  dis- 
cussed more  comprehensively  in  the  section  upon  the  differentiation  of 
pneumococcus  from  streptococcus  (page  367). 

Streptococci  are  easily  stained  by  the  usual  anilin  dyes.  Stained 
by  the  method  of  Gram,  the  pyogenic  streptococci  are  not  decolorized 
and  invariably  retain  the  gt;ntian- violet.  Certain  species  found  in  stools 
and  described  as  Gram-negative,  are  rare  and  are  non-pathogenic. 
Others  of  the  ^'Streptococcus  brevis"  variety,  and  purely  saprophytic, 
may  stain  irregularly  by  the  Gram  method. 

Cultivation. — ^The  pyogenic  streptococci  are  easily  cultivated  upon 
all  the  richer  artificial  media.  While  meat  extract-pepton  media  may 
suffice  for  certain  strains,  it  is  usually  better  to  employ  those 
media  which  have  the  beef  or  veal  infusion  for  a  basis.  For  the 
cultivation  of  more  delicate  strains  of  streptococci,  especially  when 

» V.  Lingelsheim,  "  Aetiol.  u.  Therap.  d.  Streptok.  Infek."  Beit.  z.  Exp.  Ther., 
Hft.  1,  1899. 

^  Pasquale,  Zieglers  Beit.,  xii;  Bordet,  Ann.  de  I'inst.  Pasteur,  1887;  Schottmilller, 
Munch,  med.  Woch.,  xx,  1903;  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


338  PATHOGENIC  ORGANISMS 

taken  directly  from  the  animal  or  human  body,  it  is  well  to  add  to  the 
media  animal  albumin  in  the  form  of  whole  blood,  blood  serum,  or  asci- 
tic or  pleural  transudates.  Glucose,  added  in  proportions  of  one  to  two 
per  cent,  likewise  renders  media  more  favorable  for  streptococcus  culti- 
vation. Prolonged  cultivation  of  all  races  upon  artificial  media  renders 
them  less  fastidious  as  to  cultural  requirements.  The  most  favorable 
reaction  of  media  for  streptococcus  cultivation  is  moderate  alkalinity 
(two-tenths  to  five-tenths  per  cent  alkalinity  to  phenolphthalein) . 
Growth  may  be  readily  obtained,  however,  in  neutral  media  or  even  in 
these  slightly  acid.  The  optimum  temperature  for  growth  is  at  or  about 
37.5°  C.  Above  43°  to  45°  C.,  development  ceases.  At  from  15°  to 
20°  C.,  growth,  while  not  energetic,  still  takes  place,  an  important  point 
in  the  differentiation  of  these  microorganisms  from  pneumococci.  While 
the  free  access  of  oxygen  furnishes  the  most  suitable  environment 
for  most  races  of  streptococci,  complete  anaerobiosis  does  not  pre- 
vent development  in  favorable  media.  Strictly  anaerobic  streptococci 
have  been  cultivated  from  the  human  intestinal  tract  by  Perrone  ^  and 
others. 

In  alkaline  bouillon  at  37.5°  C.,  pyogenic  streptococci  grow  rapidly, 
form  long  and  tortuous  chains,  and  have  a  tendency  to  form  flakes  which 
rapidly  sink  to  the  bottom.  Diffuse  clouding  occurs  rarely  and  is  a 
characteristic  rather  of  the  shorter  so-called  Streptococcits  brevis. 
When  sugar  has  been  added  to  the  broth  the  rapid  formation  of  lactic 
acid  soon  interferes  with  extensive  development.  This  may  be  obviated, 
especially  when  mass  cultures  are  desired,  without  sacrifice  of  the  growth- 
increasing  influence  of  the  glucose,  by  adding  to  the  sugar-broth  one  per 
cent  of  sterile  powdered  CaCOg.^ 

In  milk.  Streptococcus  pyogenes  grows  readily  with  the  formation 
of  acid,  followed,  in  most  cases,  by  coagulation  of  the  medium. 

On  agar-plates  at  37.5°  C.,  growth  appears  within  eighteen  to  twenty- 
four  hours.  The  colonies  are  small,  grayish,  and  delicately  opalescent. 
They  are  round  with  smooth  or  very  slightly  corrugated  or  lace-like 
edges,  and  rise  from  the  surface  of  the  medium  in  regular  arcs,  like 
small  droplets  of  fluid.  Microscopically  they  appear  finely  granular  and 
occasionally,  under  high  magnification,  may  be  seen  to  be  composed  of 
long  intertwining  loops  of  streptococcus  chains,  which  form  the  lace-like 
edges.  When  ascitic  fluid  or  blood  serum  has  been  added  to  agar, 
growth  is  more  energetic  and  the  colonies  correspondingly  more  rapid  in 

^  Perrone,  Ann,  de  I'inst.  Pasteur,  xix,  1905. 
2  Hiss,  Jour.  Exp.  Med.,  vi,  1905. 


STREPTOCOCCUS  PYOGENES 


339 


appearance  and  luxuriant  in  development.  In  glucose-ascitic-agar, 
acid  formation  from  the  sugar  causes  coagulation  of  albumin  with 
the  consequent  formation  of  flaky  white  precipitates  throughout  the 
medium.^ 

In  gelatin  stab-cultures  growth  takes  place  slowly,  appearing  after 
twenty-four  to  thirty-six  hours  as  a  very  thin  white  line,  or  as  discon- 
nected little  spheres  along  the  line  of  the 
stab.  The  colonies  on  gelatin  plates  are 
similar  in  form  to  those  on  agar,  but  are 
usually  more  opaque  and  more  distinctly 
white.  The  gelatin  is  not  liquefied  by  the 
pyogenic  streptococci,  though  certain  of 
the  more  saprophytic  forms  may  occa- 
sionally bring  about  slow  fluidification. 

On  Loeffler's  coagulated  blood  serum, 
growth  is  rapid  and  luxuriant,  and  may 
show  a  slight  tendency  to  confluence  if 
the  medium  is  very  moist.  Good  chain 
formation  takes  place  on  this  medium. 

Upon  potatoes,  growth  is  said  not  to 
take  place.2 

On  media  containing  red  blood  cells, 
most  pathogenic  streptococci  cause  hemolysis  and  decolorization  (see 
Fig.  74,  p.  345).  It  is  useful  to  remember  this  when  examining 
blood-culture  plates,  for  here  the  yellow  transparent  halo  of  hemo- 
lysis and  decolorization  surrounding  the  colonies  may  aid  in  differenti- 
ating these  bacteria  from  pneumococci.  This  is  of  especial  importance, 
since  many  streptococci,  when  cultivated  directly  out  of  the  human 
blood,  do  not  exhibit  chain  formation,  but  appear  as  diplococci. 

In  the  inulin-serum  media  of  Hiss,^  streptococci  do  not  produce 
acid  and  coagulation.  The  so-called  Streptococcus  mucosus,  a  capsule- 
bearing,  inulin-fermenting  microorganism,  is  very  probably  a  sub-species 
of  the  pneumococcus  (see  later  section). 

Resistance. — Streptococci  on  the  ordinary  culture  media,  without 
transplantation  and  kept  at  room  temperature,  usually  die  out  within 
ten  days  or  two  weeks.  They  may  be  kept  alive  for  much  longer  periods 
by  the  use  of  the  calcium-carbonate-glucose  bouillon,  if  the  cultures  are 

1  Libmun,  Medical  Record,  Ivii,  1900. 

*  Frosch  und  Kolle,  in  Fliigge,  "Die  Mikroorganismen,"  1891. 

'  Hiss,  Jour.  Exp.  Med.,  vi;  1905. 


Fig.  73. — Streptococcus  Col- 
onies, ON  Serum  Agar. 


340  PATHOGENIC  ORGANISMS 

thoroughly  shaken  and  the  powdered  marble  thoroughly  mixed  with 
the  bouillon  from  time  to  time.^  Preservation  at  low  temperatures 
(1°  to  2°  C),  in  the  ice  chest,  considerably  prolongs  the  Hfe  of  cul- 
tures. Virulence  is  preserved  longest  by  frequent  transplantation 
upon  albuminous  media.  In  sputum  or  animal  excreta,  streptococci 
may  remain  alive  for  several  weeks. 

Streptococci  are. killed  by  exposure  to  a  temperature  of  54°  C.  for 
ten  minutes.2  Low  temperatures,  and  even  freezing,  do  not  destroy  some 
races. 

The  action  of  various  chemical  disinfectants  has  been  thoroughly 
investigated  by  v.  Lingelsheim,^  who  reports  among  others  the  following 
results :  Carbolic  acid  1  :  200  kills  streptococci  in  fifteen  minutes.  In 
the  same  time,  bichloride  of  mercury  is  efficient  in  a  dilution  of  1  :  1,500, 
lysol  in  a  dilution  of  1  :  200,  peroxide  of  hydrogen  1  :  35,  sulphuric  acid 
1 :  150,  and  hydrochloric  acid  1  :  150.  Inhibition  is  exerted  by  car- 
bolic acid  1  :  550,  and  by  bichloride  of  mercury  1  :  65,000.  Exposure 
to  direct  sunlight  kills  streptococci  in  a  few  hours. 

Virulence  and  Pathogenicity. — Different  races  of  pyogenic  strepto- 
cocci show  considerable  variations  in  virulence,  and  there  are  few  organ- 
isms, pathogenic  both  for  animals  and  man,  which  show  such  peculiari- 
ties in  virulence.  The  character  or  severity  of  the  lesion  in  man  gives 
little  evidence  as  to  the  virulence  of  the  organism  for  animals.  Such 
differences  are,  to  a  certain  extent,  dependent  upon  inherent  individual 
characteristics,  but  are  rather  more  likely  to  be  the  consequences  of  pre- 
vious environment  or  habitat.  Prolonged  cultivation  upon  artificial 
media  usually  results  in  the  reduction  of  the  virulence  of  a  streptococcus, 
while  an  originally  low  or  reduced  virulence  may  often  be  much  en- 
hanced by  repeated  passage  of  the  streptococci  through  animals.  It  is 
noteworthy,  however,  that  while  the  passage  of  a  streptococcus  through 
rabbits  will  usually  enhance  its  virulence  for  susceptible  animals  in 
general,  repeated  passages  through  mice  may  increase  the  virulence 
for  these  animals  only,  even  occasionally  depressing  the  virulence 
for  rabbits.^ 

Among  the  donaestic  animals,  those  most  susceptible  to  experimental 
streptococcus  infection  are  white  mice  and  rabbits.     Guinea-pigs  and 

1  Hiss,  loc.  cit. 

^Sternberg,  "Textbook  of  Bact.,"  2d  ed.,  1901;  Harlmann,  Arch.  f.  Hyg.,  vii. 
3y.  Lingelsheim,   "Aetiol.  u.  Therap.  d.  Streptoc.  Inf.,"  etc.,  Beit.  z.  Exper. 
Therap.,  Hft.  1,  1899. 

^  Knorr,  Zeit.  f .  Hyg.,  xiii.  •  ' 


STREPTOCOCCUS  PYOGENES  341 

rats  are  less  easily  infected,  and  the  larger  domestic  animals,  cattle, 
horses,  goats,  cats,  and  dogs,  are  extremely  refractory.  Almost  complete 
immunity  toward  streptococcus  infections  prevails  among  birds. 

The  nature  of  the  lesions  following  animal  inoculation  depends  upon 
the  manner  of  inoculation,  the  size  of  the  dose  given,  and  most  of  all 
upon  the  grade  of  virulence  of  the  inoculated  germ.  Subcutaneous 
inoculations,  according  to  the  virulence  of  the  inoculated  material,  may 
result  in  a  simple  localized  abscess,  differing  from  a  staphylococcus 
abscess  only  in  the  more  serous  nature  of  the  exudate  and  the  frequent 
occurrence  of  edema,  or  in  a  severe  general  septicemia  with  a  hardly 
noticeable  local  lesion.-  Subcutaneous  inoculation  of  mice  results  almost 
invariably  in  general  sepsis  followed  by  death  within  thirty-six  to  forty- 
eight  hours,  or  less,  and  the  presence  of  streptococci  in  the  heart's  blood 
and  the  viscera.  Intrapleural  or  intraperitoneal  inoculation  of  suscep- 
tible  animals  with  virulent  streptococci  leads  usually  to  a  peculiarly 
hemorrhagic  form  of  exudate,  due  both  to  the  diapedesis  caused  by  the 
violent  inflammatory  process,  and  to  the  hemolysis  of  the  red  cells  by  the 
streptococcic  hemolysins.  Inoculation  of  rabbits  at  the  base  of  the  ear 
with  virulent  streptococci  may  result  in  the  formation  of  a  lesion 
indistinguishable  histologically  from  erysipelas  in  man.  ^  Marbaix  ^  has 
shown  that  such  erysipeloid  lesions  could  be  produced  in  rabbits  by 
streptococci  from  various  and  indifferent  sources,  provided  that  the 
virulence  of  each  strain  could  be  sufficiently  enhanced.  This  marked 
variability  of  the  resulting  lesion  as  determined  by  the  degree  of  virulence 
of  the  incitant,  whatever  its  original  source,  forms  a  strong  argument  in 
favor  of  the  opinion  that  all  the  pyogenic  streptococci  are  members  of  a 
single  species. 

Intravenous  inoculation  of  rabbits  with  virulent  cultures  usually 
results  in  a  rapidly  fatal  septicemia.  An  animal  which  has  died  of  a 
streptococcus  infection  usually  shows  serosanguineous  edema  about  the 
point  of  inoculation,  multiple  hemorrhagic  spots  upon  the  serous  mem- 
branes, and  congestion  of  the  viscera.  The  microorganisms  can  almost 
invariably  be  found  in  the  heart's  blood,  in  the  spleen,  and  in  the  exudate 
about  the  inoculated  area.  Microscopically,  when  the  process  has  lasted 
sufficiently  long,  parenchymatous  degeneration  of  all  the  organs  may  be 
observed.  In  the  more  chronic  infections  articular  and  periarticular 
lesions  may  occur.^ 

1  Fehldsen,  loc.  cit. ;  Frankel,  Cent,  f .  Bakt.,  vi, 

2  Marbaix,  La  Cellule,  1892. 

^Schiitz,  Zeit.  f.  Hyg.,  iii;  Hiss,  Jour.  Med.  Res.,  xix,  1908. 


342  PATHOGENIC  MICROORGANISMS 

Spontaneous  streptococcus  disease  seems  to  occur  among  some  of 
the  larger  domestic  animals.  Thus,  a  contagious  form  of  inflammation 
of  the  respiratory  passages  of  horses  has  been  attributed  to  streptococcus 
infection.^  Among  cattle  these  microorganisms  have  been  found  to 
produce  purulent  inflammation  of  the  udder  and  occasionally  post- 
partum uterine  inflammation  in  cows.  Among  the  smaller  labora- 
tory animals,  occasional  streptococcus  infections  may  be  observed  in 
rabbits.  Recently  an  epidemic  disease  among  white  mice  due  to  strep- 
tococcus was  studied  by  Kutscher.^  As  a  rule,  however,  streptococcus 
disease  is  by  far  more  rare  among  animals  than  it  is  among  human  beings. 

In  man,  a  large  variety  of  pathological  processes  may  be  caused  by 
streptococci  and  here  again  the  nature  of  the  infection,  whether  definitely 
localized  or  generally  distributed,  depends  upon  the  relationship  existing 
between  the  virulence  of  the  incitant  and  the  resistance  of  the  subject. 

The  first  cultivation  of  streptococcus  from  human  lesions  was  made 
by  Fehleisen,^  who  obtained  them  from  cases  of  erysipelas.  It  was 
long  believed  that  the  so-called  Streptococcus  erysipelatis  was  a 
similar  but  essentially  different  species  from  the  common  Streptococcus 
pyogenes.  The  production  of  erysipelas  in  animals  with  streptococci 
from  other  sources,  however,  has  shown  definitely  that  the  two  groups 
can  not  be  separated.^  Superficial  cutaneous  infections  are  frequently 
caused  by  streptococci  and  these  in  the  milder  cases  may  be  similar 
to  the  localized  abscesses  caused  by  staphylococci.  In  severe  cases, 
however,  infection  is  followed  by  rapidly  spreading  edema,  lymph- 
angitis, and  severe  systemic  manifestations  with  the  development  of  a 
grave  cellulitis,  often  threatening  life  and  requiring  energetic  surgical 
interference.  Invasion  of  the  respiratory  organs  by  streptococci  is  not 
rare,  and  may  lead  to  bronchitis,  pneumonia,  or  empyema.  They  are 
frequently  present  also  as  secondary  invaders  in  pulmonary  tubercu- 
losis.^ Streptococcus  infections  of  the  lungs  and  pleura  not  infrequently 
lead  to  pericardial  involvement. 

Suppurations  of  bone  may  be  caused  by  streptococci,  and  constitute 
a  severe  form  of  osteomyelitis.  Such  lesions  when  occurring  in  the 
mastoid  bone  are  not  infrequently  secondary  to  streptococcus  otitis  and 
may  lead  to  a  form  of  meningitis  which  is  in  most  cases  fatal.     In  the 

^  Van  de  Velde,  Monatsheft  Bakt.,  Thierheilk.,  ii. 

*  Kvischer,  Cent,  f .  Bakt.,  xlvi. 

'  Fehldsen,  loc.  cit. 

^  Marhaix,  La  Cellule,  1892;  Petruschky^  Zeit.  f.  Hyg.,  xxiii. 

6  Corned, ''Die  Tuberkulose,"  Wien,  1899. 


STREPTOCOCCUS  PYOGENES  S43 

mouth  and  throat  streptococci  may  give  rise  to  pharyngitis  and  are  one 
of  the  most  frequent  causes  of  a  form  of  tonsillitis  often  clinically  indis- 
tinguishable from  diphtheria.  The  throat  inflammation  accompanying 
scarlatina  is,  almost  without  exception,  referable  to  streptococcus  infec- 
tion.^ The  occasional  presence  of  the  streptococcus  in  the  blood  of 
scarlatina  patients,  moreover,  has  led  some  authors  to  suggest  a  pos- 
sible etiological  connection  between  this  microorganism  and  the  disease. ^ 
This,  however,  is  at  present  merely  conjectural. 

In  diphtheric  inflammations  of  the  throat,  a  secondary  streptococcus 
infection  is  a  frequent  and  serious  complication.  As  incitants  of  disease 
of  the  intestines,  streptococci  have  been  found  in  appendicular  abscesses  ^ 
and  have  been  described  as  the  cause  of  some  forms  of  infantile  diarrhea."* 
From  any  of  the  local  processes  streptococci  may  pass  into  the  circulation, 
causing  sepsis.  The  septicemia  occurring  during  the  puerperium  is  most 
often  caused  by  this  microorganism. 

Secondary  foci  in  the  viscera  may  be  established,  leading  to  pyemia,^ 
or,  if  these  localizations  occur  upon  the  heart  valves,  septic  endocarditis 
may  ensue.  All  such  forms  of  general  streptococcus  infection,  whether 
running  acute  or  chronic  courses,  present  a  high  rate  of  mortality.  The 
diagnosis  in  these  cases  is  usually  easy  if  blood  cultures  are  taken  upon 
suitable  media. 

Streptococcus  throat  infections  have  recently  appeared  in  fulmi- 
nating epidemics.  Several  small  epidemics  were  described  in  England, 
and  three  extensive  outbreaks  have  occurred  in  this  country;  one  in 
Boston  of  1,400  cases;  a  second  in  Baltimore  of  about  1,000  cases,  and 
a  third  in  Chicago  of  about  10,000  cases.  These  outbreaks  were  studied 
by  Winslow,  Stokes,  Davis,®  and  by  Rosenow.^  In  each  case  the  major- 
ity of  infections  were  traced  to  a  single  milk  supply,  though  secondary 
cases  doubtless  occurred  by  contact.  Severe  complications  such  as 
suppurative  adenitis,  otitis,  erysipelas,  peritonitis,  and  septicemia 
were  not  uncommon.  A  similar  organism — a  capsulated,  hemolytic 
streptococcus — was  found  in  each  epidemic. 

1  Baginsky,  Deut.  med.  Zeit.,  1900. 

2  Baginsky  und  Sommerfeld,  Berl.  klin.  Woch.,  xxvii,  1900. 
'  Kelly,  "Pathogenesis  of  Appendicitis." 

*Lanz  and  Tavel,  Rev.  de  Chir.,  1904;  Perrone,  Ann.  de  Tinst.  Pasteur,  1905; 
Escherich,  Jahrb.  f,  Kinderheilkunde,  1899. 
^  Lihman,  Cent.  f.  Bakt.,  xxii. 
8  Cited  from  Capps,  Jour.  A.  M.  A.,  1912,  p.  1848. 
'  Bosenow,  Jour,  of  Inf.  Dis.,  1912. 


344  PATHOGENIC  MICROORGANISMS 

Toxic  Products. — In  spite  of  extensive  researches  by  many  inves- 
tigators upon  the  nature  of  the  poisons  produced  by  streptococci, 
our  understanding  of  these  substances  is  still  very  incomplete.  The 
grave  systemic  symptoms  so  often  accompanying  comparatively 
slight  streptococcus  lesions  argue  strongly  for  the  production  by  these 
microorganisms  of  a  powerful  diffusible  poison.  Toxic  filtrates  of 
streptococcus  cultures  have  indeed  been  obtained  by  Roger/  Marmier,^ 
Baginsky  and  Sommerfeld,^  Marmorek,^  and  many  others;  but  these 
have  in  no  case  been  comparable  in  potency  to  the  soluble  toxins  of 
diphtheria  or  of  tetanus.  When  injected  into  young  guinea-pigs  in 
sufficient  quantity,  these  filtrates  produce  rapid  collapse  and  death. 
The  inability  to  produce  strong  toxins  is  generally  attributed  to  the 
difficulty  of  obtaining  very  abundant  growth  of  these  bacteria  upon 
fluid  media,  development  being  self-hmiting,  either  because  of  the  ex- 
haustion of  specific  nutritive  material  (Marmorek  ^) ,  or,  more  probably, 
because  of  the  inhibitory  effects  of  the  products  of  growth,  chiefly  acid 
formation.  This  last  factor  can  be  partially  overcome  by  the  use  of  the 
glucose-calcium-carbonate  broth  mentioned  above,  in  which  acid  neutral- 
ization is  constantly  taking  place.  For  toxin  production,  Baginsky  and 
Sommerfeld  ^  advise  a  strongly  alkaline  reaction  of  the  media;  Mar- 
morek ^  has  used  human  blood-serum-bouillon  with  success.  The 
toxins  so  produced  are  relatively  thermostable.  According  to  v. 
Lingelsheim,  heating  to  60°  or  70°  C.  destroys  them  in  part  only. 
The  endotoxins  contained  within  the  cell-bodies  of  streptococci  them- 
selves have  been  found  to  possess  but  slight  toxic  qualities. 

Apart  from  these  substances,  some  streptococci  produce  a  hemolysin 
which  has  the  power  of  bringing  about  destruction  of  red  blood  cor- 
puscles. The  observation  of  this  phenomenon  for  streptococci  was  first 
made  by  Marmorek  ^  in  1895.  According  to  this  author,  there  is  a 
direct  relationship  between  virulence  and  hemolytic  power*  Other 
investigators,  however,  notably  Schottmiiller,^  believe  the  hemolytic 
power  to  be  a  constant  characteristic  of  certain  strains  unchangeable  by 

1  Roger,  Rev.  de  med.,  1892. 

2  Marmier,  Ann.  de  I'inst.  Pasteur,  ix,  1895,  p.  533. 
'  Baginsky  und  Sommerfeld,  Berl.  klin.  Woch.,  1900. 

*  Marmorek,  Berl.  klin.  Woch.,  1902. 

^  Marmorek,  Berl.  klin.  Woch.,  xiv,  1902. 

®  Loc.  cit. 

^  Marmorek,  Ann.  de  I'inst.  Pasteur,  1895. 

*  Marmorek,  Ann.  de  I'inst.  Pasteur,  1895. 
9  Schottmuller,  Miinch.  med.  Woch.,  1903. 


STREPTOCOCCUS  PYOGENES 


345 


experimental  enhancement  or  reduction  of  the  virulence.  Streptococcus 
hemolysins  may  be  conveniently  observed  by  cultivation  of  the  organ- 
isms upon  blood-agar  plates.  They  may  be  produced  in  alkaline  pepton- 
broth  and  obtained  separate  from  the  bacteria  by  filtration — a  procedure, 
however,  in  which  the  quantities  obtained  are  never  large.  Besredka  ^ 
and  Schlesinger  ^  believe,  for  this 
reason,  that  the  hemolytic  sub- 
stances are  closely  attached  to  the 
bacterial  bodies.  The  last-named 
author,  furthermore,  has  deter- 
mined that,  in  contradistinction  to 
the  other  toxic  substances,  strepto- 
coccus hemolysins  are  extremely 
labile,  disappearing  from  culture 
fluids  after  standing  for  from  five 
to  seven  days  at  ordinary  room 
temperature. 

Immunization. — For  reasons  not 
wholly  understood  at  present,  re- 
covery from  streptococcus  infection 
does  not  to  any  marked  degree 
produce  immunity  against  these 
bacteria.     Active    immunity   may, 

however,  be  produced  in  rabbits,  goats,  horses,  and  other  domestic 
animals  by  treatment  with  gradually  increasing  doses  of  streptococcus 
cultures.^ 

In  carrying  out  such  immunizations  it  is  necessary  to  use  for  the  first 
injection  attenuated  or  dead  bacteria.  Attenuation  may  be  accom- 
plished by  moderate  heating  or  by  the  addition  of  chemicals  (terchloride 
of  iodin).  Neufeld  "^  advises,  for  the  first  injection  in  immunizing 
rabbits,  the  use  of  ascitic-broth  cultures  killed  by  heating  to  70°  C.  This 
is  followed,  after  ten  days,  by  a  second  injection  of  a  small  quantity  of 
fully  virulent  cocci.  Following  this,  injections  are  made  at  intervals 
of  ten  days  with  constantly  increasing  doses.  Modifications  of,  these 
general  principles  are  employed  in  most  laboratories. 

The  sera  of  animals  so  treated  contain  no  demonstrable  antitoxic  or 


Fig.  74. — Streptococcus  Colonies 
FROM  Blood  Culture  on  Blood- 
Agar  Plate.  Showing  areas  of  hemol- 
ysis about  colonies. 


^Besredka,  Ann.  de  Tinst.  Pasteur,  xv,  1901,  p.  880. 

2  Schlesinger,  Zeit.  f.  Hyg.,  xxiv,  1903. 

3  Koch  und  Petruschky,  Zeit.  f .  Hyg.,  xxiii,  1896. 
*  Neufeld,  Zeit.  f.  Hyg.,  xliv,  1903. 


23 


S46  PATHOGENIC  MICROORGANISMS 

antihemolytic  substances.^  They  exert,  however,  demonstrable  bacteri- 
cidal power  both  in  vivo  and  in  vitro  and  distinctly  enhance  phagocytosis 
when  brought  into  contact  with  leucocytes  and  streptococci.  This 
"opsonic"  power  has  been  noticed  both  intraperitoneally  ^  and  in  vitro. ^ 

The  protective  value  of  streptococcus  immune  sera  for  infected 
animals  is  considerable,  reaching  often  a  potency  hardly  explicable  by 
the  demonstrable  bactericidal  or  opsonic  power,  and  thereby  suggesting 
some  other  active  factor  not  understood  as  yet.*  Aronson  ^  has  produced 
immune  sera  by  the  treatment  of  horses  with  a  streptococcus  derived 
from  a  ease  of  scarlatina,  0.0004  c.c.  of  which  sufficed  to  protect  mice 
from  ten  times  the  fatal  dose  of  a  streptococcus  culture.  These  high 
protective  values,  however,  are  obtained  only  when  the  serum  injections 
are  given  simultaneously  with  the  bacteria.  Given  four  or  six  hours 
after  infection,  much  higher  dosage  must  be  employed  and  protective 
results  are  much  less  regular  in  occurrence.^  Other  antistreptococcic 
sera  have  been  produced  by  Denys,  Menger,  Tavel,  and  others,  all  show- 
ing more  or  less  marked  potency  in  protecting  animals.^ 

Since  these  sera,  while  in  a  general  way  potent  against  all  streptococci, 
have  been  found  protective  chiefly  against  the  specific  microorganism  em- 
ployed for  their  production.  Van  de  Velde,^  Denys,  Aronson,  and  others 
have  advised  the  immunization  of  the  animal  with  a  large  variety  of 
streptococcus  races,  derived  from  many  different  human  sources.  The 
resulting  "polyvalent"  serum  is  more  apt  to  exert  equally  high  protective 
powers  against  all  streptococcus  infections.  The  therapeutic  value  of 
such  sera  in  the  treatment  of  human  infections  is  still  suh  jiidice.  Un- 
deniably favorable  reports  are  published  each  year  in  increasing  number, 
but  are  by  no  means  regular  or  comparable  to  the  results  obtained  in 
diphtheria  with  diphtheria  antitoxin.  Nevertheless,  in  mild  cases  or  in 
those  in  which  the  lesions  have  been  distinctly  localized,  the  sera  seemed 
to  be  sufficiently  useful  to  justify  their  use  and  necessitate  their  stand- 
ardization. 

1  Ldngelsheim,  Zeit.  f.  Hyg.,  x,  1891.  ^  Bordet,  Ann.  de  I'lnst.  Pasteur,  1897. 

2  Denys  et  Leclef,  Cellule,  t.  ix. 

^  Benys  et  Marchand,  "Mecanisme  de  Timmunit^,"  etc.,  Brussels,  1896. 

^  Aronson,  Berl.  klin.  Woch.,  xxxii,  1896;  ibid.,  xlii  and  xliii,  1902;  ibid.,  viii  and 
ix,  1905. 

^  Denys,  "Le  S6rum  antistreptoc,"  Louvain,  1896;  Van  de  Velde,  Ann.  de  I'lnst. 
Pasteur,  1896. 

7  Denys  et  Marchand,  Bull,  de  I'acad.  roy.  de  m^d.  de  Belgique,  1898;  Menger, 
Berl.  klin.  Woch.,  1902;  Tavel,  Corr.-Bl.  f.  Schw.  Aerzte. 

8  Van  de  Velde,  Arch,  de  m€d.  exp^r.,  1897. 


STREPTOCOCCUS  PYOGENES  347 

Standardization  is  accomplished  by  the  methods  first  devised  by 
Marx  ^  for  the  standardization  of  swine-plague  serum,  and  depends  upon 
the  ability  of  the  serum  to  protect  animals  against  a  measured  dose  of 
virulent  streptococci.  Aronson^  designates  as  a  "normal  serum"  one 
of  which  0.01  c.c.  will  protect  a  mouse  against  ten  to  one  hundred 
times  the  fatal  dose  of  virulent  streptococci.  One  c.c.  of  this  serum 
equals  one  serum  unit.  Comparisons  by  animal  experiment  with  this 
standard  serum  approximately  determine  the  value  of  other  sera. 

Leucocyte  extracts  ^  have  been  employed  in  various  forms  of  strep- 
tococcus infections  of  man,  with  success  in  many  cases.  Favorable  re- 
sults have  been  obtained  with  these  extracts  in  cases  of  erysipelas. 

The  agglutinins  found  in  streptococcus  immune  sera  are  usually  most 
active  toward  the  race  of  bacteria  employed  in  the  immunization.  Other 
streptococci  are  also  agglutinated,  but  in  relatively  higher  concentra- 
tion. While  a  specific  group  reaction  is  useful  in  differentiating  strep- 
tococci from  other  species,  agglutination  can  not  be  relied  upon  to 
differentiate  individual  streptococci  from  one  another  (Hiss).  It  has 
been  found  that  a  serum  produced  with  a  streptococcus  from  one  source 
contained  a  higher  agglutinating  value  for  some  other  streptococcus 
than  for  the  one  employed  in  its  production.  Agglutinins  may  be  pro- 
duced by  treating  animals  with  dead  as  well  as  with  the  living  strep- 
tococci. While  the  technique  of  streptococcus  agglutination  is  not  diffi- 
cult when  we  are  dealing  with  strains  which  grow  with  even  clouding 
in  fluid  media,  the  frequent  spontaneous  clumping  in  broth  cultures 
necessitates  the  use  of  a  special  technique.  The  most  simple  of  these 
methods  is  the  one  in  which  calcium-carbonate-glucose  broth  is  used  for 
cultivation.^  Growing  in  this  medium  and  thoroughly  shaken  once  a 
day,  the  streptococci  are  found  evenly  divided  in  the  supernatant  fluid 
after  the  settling  out  of  the  calcium-carbonate  powder. 

Precipitins  have  been  found  by  Aronson  ^  in  streptococcus  immune 
horse  serum. 

Classification. — Differences  in  minor  cultural .  characteristics  and  in 
virulence  of  streptococci  obtained  from  various  sources  have  given  rise 
to  discussion  as  to  the  identity  of  all  races  of  streptococci.  The  earliest 
observers  were  forced  to  abandon  their  separation  of  the  streptococci  of 

^  Marx,  Deutsche  thierarzt.  Woch.,  vi,  1901. 

2  Aronson,  Berl.  klin.  Woch..  xliii,  1902;  Otio,  Arb.  a.  d.  konigl.  Inst.,  etc.,  Frank- 
furt a.  M.,  Heft  2,  190G. 

3  Hiss,  Jour.  Med.  Res.,  xix,  1908. 

*  Hiss,  Jour.  Exp.  Med.,  vii,  1905.  ^  Aronson,  Deut.  med.  Woch.,  25,  1903. 


348  PATHOGENIC  MICROORGANISMS 

erysipelas  from  other  streptococci  because  of  the  work  of  Marbaix  *  and 
others,  who  produced  erysipelas  in  rabbits  with  streptococci  from  non- 
erysipelatous  lesions,  after  enhancement  of  their  virulence.  V.  Lingel- 
sheim  2  proposed  a  purely  morphological  differentiation  of  ^'longus" 
and  "brevis";  the  former  class  including  the  streptococci  usually  found 
in  pyogenic  lesions  with  tendency  to  form  chains  of  six  or  more  links, 
the  latter  designating  the  short-chained  varieties,  including  the  less 
virulent  streptococci.  This  classification,  however,  is  not  tenable  be- 
cause of  the  dependence  of  chain  formation  upon  reaction,  consistency, 
and  nutritive  qualities  of  the  media  employed  for  cultivation,  and 
upon  the  influence  of  animal  fluids  if  the  microorganisms  are  taken 
direct  from  lesions.  Schottmiiller,^  in  1903,  proposed  a  classification 
based  both  upon  morphology  and  the  appearance  of  cultures  upon 
human  blood  agar.  By  this  method  he  divided  streptococci  into  two 
main  groups  as  follows:  I.  Streptococcus  longus  seu  erysipelatos,  consist- 
ing of  the  most  virulent  varieties,  with  tendency  to  form  long  chains, 
and  producing  hemolysis  upon  blood  media.  II.  Streptococcus  mitior 
seu  viridans,  including  less  virulent  strains,  with  usually  shorter  chain- 
formation,  and  producing  green,  non-hemolyzing  colonies  upon  blood 
media.  A  third  group.  Streptococcus  mucosus,  will'receive  special  con- 
sideration in  a  separate  section,  and  is  probably  more  closely  related 
to  the  pneumococci.  The  "viridans"  type  is  of  great  importance  med- 
ically since  it  is  so  commonly  found  in  subacute  septic  endocar- 
ditis and  has  recently  been  associated  by  Rosenow  and  others  with 
rheumatism. 

Attempts  to  separate  the  streptococci  into  subdivisions  by  their 
powers  to  ferment  various  carbohydrates  have  been  made  by  Hiss, 
Gordon,  and  others.  These  attempts  have,  so  far,  been  without  practical 
result.  Hiss  ^  indicated  a  tentative  division  of  streptococci  into  those 
which  fermented  monosaccharids  alone,  those  which  were  also  able  to 
ferment  disaccharids,  and  those  in  which  the  fermentative  powers  were 
extended  to  the  polys^ccharids,  starch,  dextrin,  and  glycogen. 

Gordon  ^  found  ten  different  fermentation  reactions  among  twenty 
pyogenic  streptococci  examined,  and  forty-eight  different  fermentation 
reactions  among  two  hundred  streptococci  isolated  from  saliva.    Other 

*  Marbaix,  loc.  cit. 

^v.  Lingelsheim,  "Aetiol.  u.  Therap.  d.  Streptokok.  Krankh.,"  etc.,  Berlin,  1899. 

^  Schottmuller,  Munch,  med.  Woch.,  1903. 

*Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exp.  Med.,  vi,  1905. 

6  Gordon,  Annual  Report,  Local  Govern.  Board,  33,  London,  1903. 


STREPTOCOCCUS  PYOGENES  349 

work  by  Andrewes  and  Horder  and  by  Buerger  ^  confirms  the  irregu- 
larity of  the  fermentation  reactions  within  this  group. 

Andrewes  and  Horder  suggest  the  following  classification: 

(1)  Streptococcus  pyogenes.  Grows  in  long  chains  and  ferments  lactose, 
saccharose,  and  salicin;  does  not  coagulate  milk.  Streptococci  which  cause 
suppurative  lesions  or  severe  systemic  infections  belong  to  this  group. 

(2)  Streptococcus  mitis.  A  saprophytic  type  found  frequently  in  the  mouth 
which  shows  the  same  cultural  characteristics  as  the  streptococcus  pyogenes, 
but  grows  in  short  chains. 

(3)  Streptococcus  anginosus.  Found  frequently  in  throats  of  scarlet-fever 
patients  which  differs  from  the  pyogenes  only  in  coagulating  milk. 

(4)  Streptococcus  salivarius.  A  short-chain  type  which  ferments  lactose, 
saccharose,  and  raffinose,  and  coagulates  milk.  Streptococci  of  this  type  are 
found  frequently  in  the  mouth,  but  are  rarely  pathogenic. 

(5)  Streptococcus  fecalis.  A  short-chain  type  which  ferments  lactose,  sac- 
charose, and  mannite.  This  type  is  foimd  normally  in  the  intestine,  and  is 
occasionally  pathogenic. 

(6)  Streptococcus  equinus.  A  short-chain  type  which  does  not  ferment  lac- 
tose.   Found  in  horse  dung  and  never  pathogenic. 

Quantitative  determinations  of  the  amount  of  acid  formed  in  vari- 
ous sugars  by  different  races  have  also  been  made  by  Winslow  and 
Palmer  ^  and  others,  but  have  led  to  no  satisfactory  classification. 

Studies  by  Hopkins  and  Lang  seem  to  show  that  the  streptococci 
found  in  most  human  infections  may  be  differentiated  from  the  ordinary 
saprophytic  types  by  the  fact  that  they  ferment  lactose  and  salicin, 
but  fail  to  ferment  raffinose,  inulin,  or  mannite.  According  to  their  re- 
sults, the  usual  saprophytic  types  found  in  the  mouth  either  fail  to  fer- 
ment saUcin  or  ferment  raffinose  or  inulin,  whereas  the  usual  fecal  types 
ferment  mannite.  They  also  found  in  infection  mannite  fermenters 
which  were  apparently  of  fecal  origin.  Streptococci  which  gave  the 
same  fermentative  reaction  as  the  mouth  saprophytes  were,  however, 
frequently  found  in  malignant  endocarditis. 

Probably  the  most  reliable  method  of  determining  the  interrelation- 
ships existing  between, bacteria,  not  only  within  this  group,  but  in  all 
bacterial  classes,  is  that  depending  upon  their  reactions  to  immune 
sera.  The  work  of  Aronson,^  Marmorek,^  and  others  has  shown  that 
streptococcus  immune  sera  produced  with  any  one  race  of  pyogenic 

1  Andrewes  and  Harder,  Lancet,  1906;  Buerger,  Jour.  Exp.  Med.,  ix,  1907. 

2  Jour,  of  Inf.  Dis.,  No.  viii,  1910,  1. 

3  Ar(ms(m,  Berl.  klin.  Woch.,  1902;  ibid.,  1903.     *  Marmarek,  Berl.  klin.  Woch.,  1902. 


350  PATHOGENIC  MICROORGANISMS 

streptococci  exerted  considerable,  though  variable,  protective  action 
against  many  other  strains  of  streptococci.  The  same  authors,  as  well 
as  many  others,  working  with  the  agglutination  reaction,  have  shown 
that  the  agglutinins  produced  with  one  streptococcus  strain  were  active 
against  many  other  streptococci.  While  most  active  usually  against 
the  particular  microorganism  with  which  they  were  produced,  this  was 
by  no  means  the  rule,  a  serum  produced  with  a  streptococcus  from  a  case 
of  sepsis,  in  one  case,  agglutinating  a  streptococcus  from  a  case  of  scar- 
latina more  highly  than  its  own  microorganism.  As  with  other  "group 
agglutinations,'^  the  more  highly  immune  the  serum  is,  the  more  gen- 
eral is  the  agglutinating  power  over  the  whole  group.  Thus,  while 
agglutination  is  practically  useless  in  separating  streptococci  from  one 
another,  it  is  highly  useful  in  differentiating  these  organisms  from  allied 
groups,  such  as  the  pneumococci.  The  immune  reactions,  therefore, 
seem  to  indicate  a  very  close  relationship  between  streptocococci  as  a  class. 

Pneumococcus  (streptococcus)  mucosus. — First  definitely  de- 
scribed by  Howard  and  Perkins  ^  in  1901,  and  subsequently  carefully 
studied  by  Schottmuller,^  who  isolated  it  from  cases  of  parametritis, 
peritonitis,  meningitis,  and  phlebitis.  It  has  since  been  described  by 
many  as  the  incitant  of  lobar  pneumonia  and  of  a  variety  of  other  le- 
sions and  as  an  apparently  harmless  inhabitant  of  the  normaL  mouth. 
Morphologically,  though  showing  a  marked  tendency  to  form  chains,  on 
solid  media  it  often  appears  in  the  diplococcus  form.  It  is  enclosed  in 
an  extensive  capsule,  which  appears  with  much  regularity  and  persist- 
ence. Though  very  similar  in  appearance,  therefore,  to  pneumococci, 
these  bacteria  do  not  appear  in  the  typical  lancet  shape.  Upon  solid 
media  they  show  a  tendency  to  grow  in  transparent  moist  masses.  The 
regularity  with  which  this  microorganism  ferments  inulin  medium, 
make  it  probable  that  it  is  more  accurate  to  place  it  with  the  group 
of  pneumococci  than  with  that  of  streptococci.^ 

Most  of  the  organisms  of  this  group  show  the  common  characteris- 
tics of  the  pneumococci  and  are  soluble  in  bile.  Occasional  strains, 
such  as  one  studied  by  Dochez  and  Gillespie,  neither  ferment  inulin 
nor  are  bile  soluble.  Rarely,  too,  does  it  cause  hemolysis.  From  the 
various  studies  carried  out  upon  this  group  it  must  be  concluded  that 
while  perfectly  distinct  in  its  formation  of  a  heavy  mucoid  colony  and 
capsulation,  this  group  is  more  closely  related  to  the  pneumococci  than  to 

^  Howard  and  Perkins,  Jour.  Med.  Res.,  1901,  N.  S.,  i. 

2  Schottmuller,  Munch,  med.  Woch.,  xxi,  1903. 

3  Hiss,  Jour.  Exp.  Med.,  1905;  Buerger,  Cent.  f.  Bakt.,  I,  xli,  1906. 


STREPTOCOCCUS  MUCOSUS  351 

the  true  streptococci.  As  would  be  expected  from  its  capsulation,  its 
virulence  is  very  powerful  and  serum  reactions  are  not  easily  carried 
out.  A  further  discussion,  of  the  immune  serum  reactions  with  this 
organism  is  included  in  the  chapter  on  pneumococci,  page  363. 

STREPTOCOCCI  AND  RHEUMATISM 

In  1910  Poynton  and  Paine  ^  described  a  diplococcus  which  they 
obtained  from  eight  cases  of  acute  rheumatic  fever  and  with  which 
they  were  able  to  produce  lesions  in  rabbits  which  they  considered 
typical  of  rheumatism.  The  organism  was  recovered  from  the  blood, 
the  pericardial  fluid,  or  the  tonsil  of  their  patients.  They  described  a 
minute  Gram-negative  diplococcus  growing  best  in  acid  media  and 
under  anaerobic  conditions,  but  capable  of  growth  on  the  surface  of  ordi- 
nary media.  Many  investigators  have  attempted  to  confirm  their  work, 
but  with  negative  results  for  the  most  part,  though  some  have  found 
streptococci  and  diplococci  from  rheumatic  lesions.  Recently  Rosenow  ^ 
has  reported  the  isolation  of  a  streptococcus  from  the  joints  of  seven 
cases  of  articular  rheumatism.  He  was  also  able  to  produce  non- 
suppurative arthritis,  endocarditis,  and  pericarditis  in  rabbits  with 
these  cultures.  He  describes  them  as  intermediate  in  character  between 
the  streptococcus  viridans  and  streptococcus  hemolyticus. 

More  recently  Rosenow^  has  reported  the  production  of  gastric 
ulcers  in  rabbits  and  dogs  with  streptococci  of  a  certain  grade  of  viru- 
lence. He  has  also  obtained  streptococci  from  human  peptic  ulcers 
which  showed  a  remarkable  ''affinity"  for  the  gastric  mucous  membranes 
of  experimental  animals. 

Rosenow  seems  inclined  to  think  on  his  later  work  that  individual 
strains  of  streptococcus  may  acquire  predilections  for  definite  tissues, 
consequently  causing  rheumatic  lesions,  muscular  lesions,  lesions  in  the 
gastric  mucosa,  etc.  He  bases  this  opinion  both  on  experimental  work 
and  upon  observations  in  patients.  Judgment  upon  this  point  of  view 
must  for  the  present  be  held  in  abeyance,  yet  it  seems  to  us  more  likely 
that  specific  localization  of  streptococci,  especially  in  connection  with 
rheumatism,  may  find  an  explanation  in  the  hypersusceptibility  of  in- 
dividual tissues  to  the  organism,  such  as  that  indicated  in  the  work 
of  Faber  who  sensitized  joints  with  extracts  of  streptococci,  subse- 
quently producing  lesions  in  these  joints  by  intravenous  injections  of 
the  microorganisms  themselves. 

1  Poynton  and  Paine,  Lancet,  1900,  ii,  861,  932. 

2  Rosenow,  Jour.  A.  M.  A.,  1913,  Ix,  1223. 

3  Rosenow,  Jour.  A.  M.  A.,  1913,  Ixi,  1947,  2007. 


CHAPTER   XXIII 
DIPLOCOCCUS    PNEUMONIiE 

(Pneumococcus,  Diplococcus  lanceolatus) 

The  opinion  that  lobar  pneumonia  is  an  infectious  disease  was  held 
by  many  far-sighted  clinicians  long  before  the  actual  bacteriological 
facts  had  been  ascertained.  This  idea,  so  well  founded  upon  the  nature 
of  the  clinical  course  of  the  disease,  with  its  violent  onset  and  equally 
rapid  defervescence,  led  many  of  the  earlier  bacteriologists  to  make  it  the 
subject  of  their  investigations — a  subject  made  doubly  difficult  by  the 
abundant  bacterial  flora  found  normally  in  the  upper  respiratory  pas- 
sages, and  by  the  fact,  which  is  now  recognized,  that  lobar  and  other 
pneumonias  are  by  no  means  always  caused  by  one  and  the  same  micro- 
organisms. 

Cocci  of  various  descriptions  and  cultural  characteristics  were  isolated 
from  pneumonia  cases  by  Klebs,^  Koch,^  Giinther,^  Talamon,^  and  many 
others,  which,  however,  owing  to  the  insufficient  differential  methods  at 
the  command  of  these  investigators,  can  not  positively  be  identified 
with  the  microorganism  now  known  to  us  as  Diplococcus  pneumoniae 
or  the  pneumococcus.  Although  thus  unsuccessful  as  to  their  initial 
object,  these  early  investigations  were  by  no  means  futile,  in  that  they 
gave  valuable  information  regarding  the  manifold  bacterial  factors 
involved  in  acute  pulmonary  disease  and  incidentally  led  to  the  dis- 
covery by  Friedlander  ^  of  B.  mucosus  capsulatus. 

Communications  upon  lance-shaped  cocci  found  in  saliva,  and 
capable  of  producing  septicemia  in  rabbits,  were  published  almost  simul- 
taneously by  Sternberg  ^  and  by  Pasteur  ^  in    1880.     These   workers 

^Klehs,  Arch.  f.  exp.  Path.,  1873. 
^Koch,  Mitt.  a.  d.  kais.  Gesundheitsamt,  Bd.  1. 
^Gunther,  Deut.  med.  Woch.,  1882. 
•»  Talamon,  Progr.  mM.,  1883. 
^Friedlander,  Virchow's  Arch.,  Ixxxvii. 
« Sternberg,  Nat.  Board  of  Health  Bull.,  1881. 
» Pasteur,  Bull,  de  Tacad.  de  med.,  1881. 
352 


DIPLOCOCCUS  PNEUMONIA  353 

beyond  reasonable  doubt  were  dealing  with  the  true  pneumococcus,  but 
did  not  in  any  way  associate  the  microorganisms  they  described  with 
lobar  pneumonia.  The  solution  of  this  problem  was  reserved  for  the 
labors  of  A.  Frankel  ^  and  Weichselbaum  ^  who  published  their  results, 
independently  of  each  other,  in  1886,  demonstrating  beyond  question 
that  the  pneumococcus  is  the  etiological  factor  in  a  large  majority  of 
cases  of  lobar  pneumonia. 

Morphology  and  Staining. — The  morphology  of  the  pneumococcus  is, 
in  general,  one  of  the  most  valuable  guides  to  its  identity. 

When  typical,  the  pneumococcus  is  a  rather  large,  lancet-shaped  coc- 
cus, occurring  in  pairs,  and  surrounded  by  a  definite  and  often  wide 
capsule,  which  usually  includes  the  two  approximated  cocci  without  a 
definite  indentation  opposite  their  lines  of  division.  The  pneumococci 
may,  however,  occur  singly  or  in  short  chains,  and  even  fairly  long 
chains  are  not  infrequently  met  with  under  artificial  cultural  conditions. 
This  may  be  chiefly  due  to  the  cultural  conditions  or  may  be  a  promi- 
nent characteristic  of  certain  strains.  Apparently  the  capsules  of  or- 
ganisms making  up  the  chains  are  continuous;  wavy  indentations  are 
usually  present,  however,  in  the  capsule  of  chains,  and  at  times  distinct 
divisions  are  observed. 

The  chief  variations  from  the  typical  morphology  consist  either  in 
the  assumption  of  a  more  distinctly  spherical  coccus  type,  or  in  an 
elongation  approximating  the  bacillary  form.  Under  certain  conditions 
of  artificial  cultivation  a  distinct  flattening  of  the  organisms,  particularly 
of  those  making  up  chains,  may  be  seen,  and  even  the  impression  of  a 
longitudinal  line  of  division,  characteristic  of  many  streptococcus 
cultures,  is  not  infrequently  gained. 

The  capsules  under  certain  conditions,  especially  in  artificial  media, 
may  be  absent  or  not  demonstrable,  and  in  certain  strains  capsules  ap- 
parently may  not  be  present  under  any  conditions.  Practically  any  of 
the  described  variations  may  dominate  one  and  the  same  culture  under 
different  or  even  apparently  the  same  conditions  of  cultivation,  and  all 
grades  may  occur  in  capsule  development,  from  its  typical  formation 
through  all  variations,  to  its  total  and  apparently  permanent  absence. 

The  presence  or  absence  of  capsules  depends,  to  a  large  extent,  upon 
the  previous  environment  of  the  pneumococci  under  observation.  The 
most  favorable  conditions  for  the  development  or  preservation  of  the 
pneumococcus  capsule  are  found  in  the  body  fluids  of  man  and  animals 

1  A.  Frankel,  Zeit.  f.  klin.  Med.,  x,  1886. 

2  Weichselbaum,  Med.  Jahrbticher,  Wien,  1886. 


354  PATHOGENIC  MICROORGANISMS 

suffering  from  pneumococcus  infection.  For  instance,  capsules  may  be 
demonstrated  with  ease  by  the  usual  capsule-staining  methods  in  the 
blood,  serum,  and  inflammatory  exudate  of  the  infected  rabbit  and 
white  mouse.  Capsules  may  be  equally  well  marked  in  the  fresh  sputum 
of  pneumonia  patients,  especially  in  the  early  stages  of  the  disease  and 
in  the  exudate  accompanying  such  pneumococcus  infections  as  menin- 
gitis, otitis  media,  and  empyema.  In  sputum  and  the*  exudates  of 
various  localized  infections,  the  organisms  are,  however,  frequently 
degenerated  or  under  chemical  conditions  unfavorable  for  capsule 
staining,   and  satisfactory  results  are  not  then  easily  obtained.     The 


Fig.  75. — Pneumococci,  Grown  on  Fig.  7G. — Pneumococct,  from  Rab- 

Loeffler's     Serum.      (Capsule     stain  bit's  Heart  Blood.     (Capsule  stain  by 

by     gentian-violet-potassium-carbonate  copper-sulphate  method.) 
method.) 

same  is  often  true  of  the  scrapings  from  lungs  of  patients  dead  of 
pneumonia,  even  in  the  stage  of  red  hepatization. 

In  artificial  cultivation,  if  the  nutrient  medium  is  not  milk  or  does  not 
contain  serum,  capsules  can  not  usually  be  demonstrated  by  the  ordinary 
methods  of  preparing  and  staining.  Capsules  may,  however,  with  much 
regularity  be  demonstrated  on  pneumococci,  in  agar,  broth,  or  on  almost 
all,  if  not  all,  artificial  media,  irrespective  of  the  length  of  time  the  organ- 
isms have  been  under  artificial  cultivation  if  beef  or  rabbit  serum  is  used 
as  the  diluent,  when  they  are  spread  on  the  cover-glass  for  staining.^ 

The  pneumococcus  is  non-motile  and  possesses  no  flagella.  Spores 
are  not  formed.  Swollen  and  irregular  involution  forms  are  common 
in  cultures  more  than  a  day  old. 

» Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exp.  Med.,  vi,  1905. 


DIPLOCOCCUS  PNEUMONIiE  355 

The  pneumococcus  is  stained  readily  with  all  the  usual  aqueous 
anilin  dyes.  Stained  by  the  method  of  Gram,  it  is  not  decolorized. 
Special  methods  of  staining  have  been  devised  for  demonstra- 
tion of  the  capsule.  The  ones  most  generally  used  are  the  glacial 
acetic-acid  method  of  Welch  ^  and  the  copper-sulphate  method  of  Hiss.^ 
More  recently  Buerger^  has  devised  a  more  complicated  method  for 
staining  capsules,  for  which  he  claims  differential  value.  (For  methods 
see  section  on  Technique,  p.  98.) 

For  simple  staining  of  pneumococci  in  tissue  sections,  the  Gram- 
Weigert  technique  is  excellent.  For  demonstration  of  the  capsules 
in  tissue  sections,  Wadsworth  *  has  described  a  simple  method. 

Cultivation  and  Isolation. — The  pneumococcus  being  more  strictly 
parasitic  than  many  other  bacteria,  presents  greater  difficulties  in  its 
cultivation.  On  meat-extract  media  growth  does  not  take  place  with 
regularity.  On  those  media,  however,  which  have  beef  or  veal  infusion 
for  their  basis,  growth  can  be  obtained  with  considerable  regularity, 
although  such  growth  may  be  sparse  and  delicate. 

Growth  takes  place  most  regularly  at  a  temperature  of  37.5°  C. 
Development  does  not  usually  occur  below  25°  nor  above  41°  C.^  At 
ordinary  room  temperature,  18-22°  C,  the  temperature  used  for  gelatin 
cultivation,  growth  either  does  not  take  place  at  all  or  is  exceedingly 
slow  and  unenergetic. 

Aerobic  and  anaerobic  conditions  both  permit  the  growth  of 
pneumococcus,  there  being  very  little  difference  in  speed  or  extent 
of  growth  along  the  course  of  deep  stab  cultures  in  favorable  media. 
The  most  favorable  reaction  of  media  for  the  cultivation  of  this  micro- 
organism is  neutrality  or  very  slight  alkahnity.  Slight  acidity,  how- 
ever, if  not  exceeding  eight-tenths  per  cent,  does  not  materially  hamper 
development. 

The  growth  of  pneumococci  on  all  media  may  be  considerably 
enhanced  by  the  addition  to  these  media  of  animal  or  human  serum  or 
whole  blood.  Additional  substances  which,  among  others,  unquestion- 
ably have  a  favorable  influence  upon  pneumococcus  growth,  are  glucose, 
nutrose,  and  glycerin.  The  addition  of  the  latter  substances  to  the 
media,  however,  probably  because  of  acid  formation,  hastens  the  death 

1  Welch,  Johns  Hopk.  Hosp.  Bull,  xiii,  1892. 

«  Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exp.  Med.,  vi,  1905. 

^Buerger,  Medical  News,  Ixxxviii,  1904. 

*  Wadsworth,  "  Studies  by  the  Pupils  of  W.  T.  Sedgwick,"  Chicago,  1896, 

•A.  Frankel,  Dent.  med.  Woch.,  xiii,  1886. 


356  PATHOGENIC   MICROORGANISMS 

of  pneumococcus  cultures.  An  increase  of  the  amount  of  pepton 
used  for  the  preparation  of  media  is  desirable  for  the  cultivation  of 
this  microorganism;  two  to  four  per  cent  of  pepton  may  be  found 
advantageous. 

In  suitably  alkaline,  nutrient  broth,  growth  is  rapid,  and  within 
twenty^four  hours  leads  to  slight  clouding  of  the  fluid.  This  clouding, 
as  a  rule,  eventually  disappears  as  the  microorganisms,  sinking  to 
the  bottom  of  the  tube  or  disintegrating,  leave  the  fluid  more  or 
less  clear.  In  broth,  pneumococci  have  a  tendency  to  form  short 
chains.  When  glucose  has  been  added  to  the  broth,  growth  is  more 
rapid  and  profuse,  but  considerable  acid  formation  causes  the  cultures 
to  die  out  rapidly.  It  is  possible,  however,  to  employ  glucose  as  a 
growth-enhancing  element  in  broth  cultures  without  interfering  with  the 
viability  of  the  cultures  by  adding  small  quantities  (one  per  cent)  of 
sterile,  powdered  calcium  carbonate.  This  method  of  cultivation  in 
broth  is  especially  adapted  to  the  production  of  mass  cultures  for  purposes 
of  immunization  or  agglutination.^  The  addition  of  ascitic  fluid  or  blood 
serum  to  broth,  in  the  proportion  of  one  to  three,  makes  an  extremely 
favorable  medium  in  which  growth  is  rapid  and  profuse. 

Upon  agar  plates,  pneumococcus  growth  is  not  unlike  that  of  strepto- 
coccus. The  colonies  are  small,  round,  and  slightly , more  transparent 
than  those  of  the  streptococci.  They  appear  more  moist  than  strepto- 
coccus colonies  and  often  are  more  flat.  Microscopically  examined,  the 
colonies  are  finely  granular,  with  dark  centers  and  slightly  corrugated 
lighter-colored  peripheral  areas.  Under  high  magnification  no  such  in- 
tertwining convolutions  can  be  seen  as  those  noticed  under  similar 
magnification  in  streptococcus  cultures.  The  addition  of  animal  albu- 
min to  agar  results  in  the  more  rapid  development,  larger  size,  and  deeper 
opacity  of  the  colonies. 

Agar  stab  cultures  show  growth  within  twenty-four  to  thirty-six 
hours,  which  takes  place  with  equal  thickness  along  the  entire  course  of 
the  stab.  There  is  nothing  distinctive  in  these  cultures  to  differentiate 
them  from  similar  streptococcus  cultures. 

In  gelatin  plate  snidstab  cultures  at  22°  C,  growth,  as  a  rule,  does  not 
take  place.  This,  however,  is  not  true  of  all  races  of  pneumococci. 
Occasionally  strains  are  met  with  which  will  grow  fairly  abundantly  in 
gelatin  at  a  temperature  of  22°  C.  When  the  gelatin  is  rendered  suffi- 
ciently firm  to  bear  25°  to  26°  C.  without  melting,   growth   appears 


Hiss,  Jour.  Exp.  Med.,  vii,  1905. 


DIPLOCOCCUS  pneumonia;  357 

slowly  and  sparsely  as  minute,  grayish-white,  transparent  colonies. 
The  gelatin  is  not  liquefied  by  the  organisms. 

Growth  upon  milk  is  profuse,  resulting  in  the  production  of  acid  and 
coagulation  of  the  medium.  Races  are  encountered  in  which  this  is 
suppressed  and  coagulation  in  milk  is  absent  or  long  delayed. 

Upon  potato,  thin,  gray,  moist  growth  occurs,  hardly  visible  and  in- 
distinguishable from  an  increased  moisture  on  the  surface  of  the  medium. 

Upon  Loeffler's  coagulated  blood  serum,  the  pneumococcus  develops 
into  moist,  watery,  discrete  colonies  which  tend  to  disappear  by  a  drying 
out  of  the  colonies'  after  some  days,  differing  in  this  from  streptococcus 
colonies,  which,  though  also  discrete,  are  usually  more  opaque  and 
whiter  in  appearance  than  those  of  the  pneumococcus  and  remain  un- 
changed for  a  longer  time.  This  medium,  as  will  be  seen,  is  useful  in 
differentiating  pnexmiococci  from  the  so-called  Streptococcus  mucosus. 

Upon  mixtures  of  whole  rabbit's  blood  and  agar,  the  pneumococcus 
grows  well,  and  forms,  after  four  or  five  days,  thick  black  surface  colo- 
nies, not  unlike  sun  blisters  on  red  paint.  These  colonies  are  easily 
distinguished  from  those  of  streptococci,  and  are  of  considerable  differ- 
ential value.^ 

Pneumococcus  colonies  on  blood  plates  may  cause  a  slight  halo  of 
hemolysis  and  methemaglobin  formation  after  48  hours  or  longer  in 
the  incubator. 

Guarnieri  ^  has  recommended  a  medium  with  a  pepton-beef-infusion 
basis  rendered  semisolid  by  mixtures  of  agar  and  gelatin.  A  modifica- 
tion of  this  medium  has  been  described  by  Welch  ^  and  has  been  much 
employed.  Cultivation  within  eggs  and  upon  egg  media  ^  has  been  used. 
Wadsworth  ^  has  recommended  a  medium  composed  of  ascitic  fluid  to 
which  agar  has  been  added — sufficient  to  give  a  soft  jelly-like  consist- 
ency. He  observed  prolonged  viability  and  the  preservation  of  the 
virulence  on  this  medium. 

For  the  purpose  of  differentiating  pneumococci  from  streptococci, 
Hiss  ^  devised  a  medium  of  beef  serum  one  part,  and  distilled  water  two 
parts,  to  which  is  added  one  per  cent  of  inulin  (c.  p.),  and  enough  litmus 
to  render  the  medium  a  clear,  transparent  blue.  By  fermentation  of 
the  inulin  the  pneumococcus  acidifies  this  mixture,  causing  coagulation 
of  the  serum.    Streptococci  do  not  ferment  inulin. 

1  Hiss,  loc.  cit.  3  Welch,  Johns  Hopk.  Hosp.  Bull.,  iii,  1S92. 

2  Gvjamieri,  Att.  dell'  Acad,  di  Roma,  1883.       ■*  Sdavo,  Riv.  d'Igiene,  1894. 
B  Wadsworth,  Proc.  N.  Y.  Path.  Soc,  1903. 

•  Hissj  Jour.  Exp.  Med.,  vi,  1905. 


358  PATHOGENIC  MICROORGANISMS 

For  the  isolation  of  pneumococci  from  mixed  cultures  or  from  mate- 
rial containing  other  species,  such  as  sputum,  surface  smears  of  the 
material  are  made  upon  plates  of  neutral  glucose-agar,  glucose-serum- 
agar,  or  blood  agar.  According  to  the  number  of  bacteria  present  in 
the  infected  material,  it  may  be  smeared  directly  upon  the  plate,  or 
diluted  with  sterile  broth  before  planting.  After  incubation  for  twenty- 
four  hours,  the  pneumococcus  colonies  are  easily  differentiated  from  all 
but  those  of  streptococcus.  With  practice,  however,  they  may  be  dis- 
tinguished from  these  also,  by  their  smoother  edges  and  greater  trans- 
parency and  flatness. 

For  the  isolation  of  pneumococci  from  sputum,  the  sputum  may  be 
washed  by  gently  rinsing  in  successive  watch  glasses  containing  salt 
solution,  then  rubbed  up  in  a  glass  tube  or  mortar  with  a  little  broth 
and  injected  into  a  white  mouse,  either  into  the  base  of  the  tail  or  into 
the  peritoneum,  taking  great  care  not  to  puncture  the  liver.  If  virulent 
pneumococci  are  present,  death  will  occur  within  24  hours  or  there- 
abouts. Pneumococci  wilL  be  found  in  pure  culture  in  the  heart's 
blood,  and  in  large  amounts  in  the  peritoneal  exudate. 

Resistance. — On  artificial  media,  the  viability  of  the  pneumococcus 
is  not  great.  Cultures  upon  agar  or  bouillon  should  be  transplanted 
every  third  or  fourth  day,  if  the  cultures  are  kept  within  an  incubator. 
In  all  media  in  which  rapid  acid  formation  takes  place,  such  as  glu- 
cose media,  the  death  of  cultures  may  occur  more  rapidly.  In  media 
containing  albumin  and  of  a  proper  reaction,  preservation  for  one  or 
even  two  weeks  is  possible.  The  longer  the  particular  race  has  been 
kept  upon  artificial  media,  the  more  profuse  is  its  .growth,  and  the 
greater  its  viability,  both  qualities  going  hand  in  hand  with  diminish- 
ing parasitism.  The  length  of  life  may  be  much  increased  by  preserva- 
tion at  low  temperature,  in  the  dark,  and  by  the  exclusion  of  air.  In 
caJcium  carbonate  broth  and  kept  in  the  ice-chest,  cultures  may  often 
remain  alive  for  months. 

Neufeld  has  succeeded  in  keeping  pneumococci  alive  and  virulent, 
by  taking  out  the  spleens  of  mice  dead  of  pneumococcus  infection  and 
preserving  them  in  a  Petri  dish  in  a  dessicator,  in  the  dark  and  cold.  In 
this  way,  the  organisms  can  be  cultivated  from  the  spleen,  and  will  be 
found  virulent  for  longer  periods  than  in  culture  media. 

In  sputum  the  viability  of  pneumococci  seems  to  exceed  that  ob- 
served in  culture.    The  studies  of  Guarnieri,^  Bordoni-Uffreduzzi,^  and 

1  Guamieri,  Att.  della  R.  Acad.  Med.  di  Roma,  iv,  1888. 
^  Bordm^'TIffrediLZzif  Arch.  p.  1.  so.  med.,  xv»  1891. 


DIPLOCOCCUS  PNEUMONIiE  359 

others  have  shown  that  pneumococci  slowly  dried  in  sputum  may  re- 
main alive  and  virulent  for  1  to  4  months,  when  protected  from  light; 
and  as  long  as  nineteen  days  when  exposed  to  diffused  light  at  room 
temperature.  Experiments  by  Ottolenghi  ^  have  confirmed  these  re- 
sults; the  virulence  seems,  in  Ottolenghi's  experiments,  to  have  become 
considerably  attenuated  before  death  of  the  cocci.  Recent  studies  by 
Wood,2  whose  attention  was  focused  chiefly  upon  pneumococcus  viability 
in  finely  divided  sputum — in  a  condition  in  which  inhalation  transmission 
would  be  possible — have  shown  that  pneumococci  survive  for  only  about 
one  and  one-half  hours,  under  ordinary  conditions  of  light  and  tempera- 
ture.    Exposed  to  strong  sunlight  pneumococci  die  off  within  an  hour. 

Low  temperatures  slightly  above  zero  are  conducive  to  the  pro- 
longation of  life  and  the  preservation  of  virulence. 

The  resistance  of  the  pneumococcus  to  heat  is  low,  52°  C.  destroying  it 
in  ten  minutes.^  To  germicidal  agents,  carbolic  acid,  bichlorid  of  mer- 
cury, permanganate  of  potassium,  etc.,  the  pneumococcus  is  sensitive, 
being  destroyed  by  weak  solutions  after  short  exposures. 

The  disinfection  of  sputum,  difficult  because  of  the  protective  coat- 
ing of  the  secretions  about  the  bacteria,  has  been  recently  studied  by 
Wadsworth."*  The  conclusions  reached  by  this  writer  indicate  that 
pneumococci  in  exudates  are  most  rapidly  destroyed  by  twenty  per  cent 
alcohol,  other  and  stronger  disinfectants  being  less  efficient,  probably 
because  of  slighter  powers  of  diffusion. 

Virulence  and  Pathogenicity. — The  virulence  of  pneumococci  is  sub- 
ject to  much  variation,  depending  upon  the  length  of  time  during  which 
it  has  been  cultivated.  It  has  been  mentioned  above  that  under  condi- 
tions such  as  those  prevailing  in  dried  sputum  or  blood  ^  the  virulence  of 
pneumococci  may  be  preserved  for  several  weeks.  Ordinarily,  the 
virulence  diminishes  as  the  cocci  adapt  themselves  to  life  upon  artificial 
media.  Upon  media  containing  animal  albumin,  such  as  ascitic  fluid  or 
blood  agar,  this  attenuation  is  less  rapid  than  upon  the  simple  meat- 
infusion  preparations. 

In  the  blood  of  rabbits  dead  of  a  pneumococcus  infection,  taken 
directly  into  sterilized  tubes,  sealed  and  kept  in  the  dark,  Foa  ^  has  been 
able  to  preserve  the  virulence  of  pneumococci  for  as  long  as  forty-five 
days.  Preservation  in  the  spleen  of  animals  dead  of  pneumococcus 
infection,  as  practiced  by  Neufeld,  has  been  mentioned  above.  Whether 

1  Ottolenghi,  Cent.  f.  Bakt.,  xxv,  1889.        ^  Wood,  Jour.  Exp.  Med.,  vii,  1905. 
3  Sternberg,  Cent.  f.  Bakt.,  xii,  1891.  •*  Wadsworth,  Jour.  Inf.  Diseases,  iii,  1906. 
6  Quarnieri,  loc.  cit,  ^  Foa,  Zeit.  f .  Hyg.,  iv,  1888. 


360  PATHOGENIC  MICROORGANISMS 

or  not  the  virulence  of  pneumococci  is  attenuated  by  sojourn  within  the 
human  body  during  disease  is  uncertain.  The  attenuation  of  virulent 
pneumococci  on  artificial  media  may  be  hastened,  according  to  Frankel/ 
by  cultivation  of  the  organism  at  or  above  a  temperature  of  41°  C. 

The  virulence  of  attenuated  cultures  may  be  rapidly  enhanced  by 
passage  of  the  organisms  through  the  bodies  of  susceptible  animals. 
The  virulence  of  strains  may  be  so  enhanced  that  one  one-millionth  of 
a  c.c.  will  kill  a  mouse. 

Among  the  domestic  animals  white  mice  and  rabbits  are  most  sus- 
ceptible. Guinea-pigs,  dogs,  rats,  and  cats  are  much  more  resistant. 
Birds  are  practically  immune. 

The  results  of  pneumococcus  inoculation  into  susceptible  animals 
vary  according  to  the  size  of  the  dose,  the  virulence  of  the  introduced 
bacteria,  the  mode  of  administration,  and  the  susceptibility  of  the 
subject  of  the  inoculation.  Subcutaneous  inoculation  of  virulent 
pneumococci  into  mice  and  rabbits  usually  results  in  an  edematous 
exudation  at  the  point  of  inoculation,  which  leads  to  septicemia  and 
death  within  twenty-four  to  seventy-two  or  more  hours.  When  the 
dose  has  been  extremely  small  or  the  culture  unusually  attenuated,  a 
localized  abscess  may  be  the  only  result.  Intravenous  inoculation  is 
usually  more  rapidly  fatal  in  these  animals  than  the  subcutaneous 
method.  Intraperitoneal  inoculation  in  rabbits  results  in  the  formation 
of  a  rapidly  spreading  peritonitis  in  which  the  exudates  are  apt  to  be 
accompanied  by  a  deposit  of  fibrin,  and  to  lack  the  transparent  red  color 
so  often  caused  by  the  hemolyzing  streptococci.  With  very  virulent 
strains,  these  differences  are  less  marked.  In  almost  all  of  these  infec- 
tions death  is  preceded  by  septicemia  and  the  microorganisms  can  be 
recovered  from  the  heart's  blood  of  the  victims. 

The  production  in  animals  of  lesions  comparable  to  the  lobar  pneu- 
monia of  human  subjects  has  been  the  aim  of  many  investigators. 
Wadsworth,  ^  recognizing  that  such  lesions  probably  depended  upon  the 
partial  immunity  which  enabled  the  infected  subjects  to  localize  the 
pneumococcus  processes  in  the  lungs  after  infection  by  way  of  the 
respiratory  passages,  succeeded  in  producing  typical  lobar  pneumonia 
in  rabbits  by  partially  immunizing  these  animals  and  inoculating  them 
intratracheally  with  pneimiococci  of  varying  virulence.  Lamar  and 
Meltzer  ^  produced  lobar  pneumonia  in  dogs  in  1912  by  injecting  cul- 

1  Frdnkel,  Deut.  med.  Woch.,  13,  1886. 

2  Wadsworth,  Amer.  Jour.  Med.  Sci.,  May,  1904.  • 

3  Lamar  and  Meltzer^  Jour.  Exp.  Med.,  xv,  1912. 


DIPLOCOCCUS  PNEUMONIA  361 

tures  in  the  bronchi  and  blowing  them  into  the  finer  bronchioles  with 
air.  Similar  experiments  have  been  made  by  Wintemitz  and  Hirsch- 
felder.^ 

In  man,  the  most  frequent  lesion  produced  by  the  pneumococcus  is 
acute  lobar  pneumonia.  About  ninety  per  cent  of  all  cases  of  this 
disease  are  caused  by  the  pneumococcus,^  the  remainder  being  due  to 
streptococci,  influenza  bacilli,  Friedlander's  bacilli,  and  exceptionally 
to  other  microorganisms.  Lobular  pneumonia  is  caused  by  the  pneu- 
mococcus with  almost  equal  regularity.  Infection  in  pneumonia  prob- 
ably occurs  through  the  lung,  and  in  such  experiences  as  those  of  the 
French  and  Americans  at  Panama  and  those  of  the  English  in  the  South 
African  diamond  mines,  it  would  appear  that  pneumonia  might  be  a 
contagious  disease.  According  to  Cole,  ^  when  pneumonia  is  secondary 
to  septicemia  it  is  usually  of  the  lobular  type.  Experiments  of  Meltzer 
seem  to  indicate  that  infection  is  facilitated  by  closure  of  the  small 
bronchioles,  and  cold  or  chilling  may  possibly  stimulate  the  mucous 
glands  so  as  to  plug  these. 

In  the  discussions  given  below  concerning  the  types  of  pneumococci, 
it  will  appear  that  the  type  of  pneumococcus  found  in  the  lung  not  often 
coincides  with  that  found  in  the  mouths  of  normal  individuals.  The 
types  I,  II  and  III,  according  to  Dochez  and  Avery,^  are  found  only  in 
association  with  the  disease,  but  the  fourth  group  is  the  one  which  is 
found  in  sputum  of  many  normal  individuals.  A  person  in  contact 
with  a  pneumococcus  patient  may  possibly  carry  the  more  virulent 
groups  in  his  mouth,  thus  constituting  a  pneimiococcus  carrier.  During 
the  course  of  these  diseases  the  cocci  are  found  in  large  numbers  witljin 
the  pulmonary  alveoli,  and  in  the  capillaries  and  lymph  vessels  of  the 
lung.  Whether  or  not  the  pneumococci  enter  the  blood  stream  in  all 
these  cases  is  a  question  not  yet  definitely  settled.  Frankel  ^  states  it  as 
his  belief  that  in  most,  if  not  in  all,  cases,  the  diplococci  at  some  time 
during  the  diseases  could  be  found  in  the  circulating  blood.  Prochaska 
in  a  study  of  ten  unselected  cases  obtained  positive  blood  cultures  in 
every  one  of  them.  A  review  of  the  literature  upon  the  question  indi- 
cates positive  blood-culture  findings  in  certainly  over  twenty-five  per 
cent  of  the  cases. 

*  Wintemitz  and  Hirschfelder,  Jour.  Exp.  Med.,  xvii,  1913. 
2  better,  Compt.  rend,  de  la  soc.  de  biol.,  1890. 

.  3  Cole,  Harvey  Lecture,  New  York,  Dec.  13,  1913. 

*  Dochez  and  Avery,  Jour.  Exper.  Med.,  xxi,  1915. 
^Frankel,  "v.  Leyden  Festschr.,"  1902. 

24 


362  PATHOGENIC  MICROORGANISMS 

In  complications  of  pneumonia,  pneumococci  are  found  usually  in 
the  pleura  where  they  may  cause  a  simple  dry  pleurisy  or  even  empy- 
ema. Less  frequently  they  may  cause  pericarditis  and  endocarditis. 
Meningitis  may  be  either  secondary  to  pneumonia  or  independent. 
Such  cases  are  grave,  almost  invariably  ending  in  death.  Other  lesions 
which  may  be  caused  by  pneumococci,  either  as  post-pneumonic  proc- 
esses or  without  previous  pneumonia,  are  otitis  media,  osteomyelitis, 
and  arthritis.  Cases  of  pneumococcus  peritonitis  occur  sometimes 
secondary  to  appendicular  inflammations,  occasionally  without  traceable 
portal  of  entry.  Severe  catarrhal  conjunctivitis  may  be  caused  by  these 
diplococci,  usually  during  the  course  of  a  pneumonia.  Ulcerative 
endocarditis  with  pneumococcus  septicemia,  apparently  independent  of 
a  pulmonary  lesion,  is  not  infrequent. 

Toxic  Products  of  the  Pneumococcus. — Our  knowledge  of  pneumococ- 
cus poisons  is  still  very  imperfect.  Attempts  to  obtain  soluble  toxins  by 
the  filtration  of  cultures  have  been  practically  unsuccessful.  G.  and  F. 
Klemperer,^  Mennes,^  Pane,^  Foa  and  Carbone,*  and  others  failed  to 
obtain  pneumococcus  filtrates  of  any  degree  of  toxicity,  though  working- 
with  highly  virulent  strains.  The  feeble  toxin  so  obtained  produced  ii 
antitoxin. 

The  general  failure  to  procure  strong  soluble  poisons  from  cultures, 
gives  weight  to  the  a-ssumption  that  the  most  potent  toxic  products  of 
pneumococci  are  in  the  nature  of  endotoxins  and  closely  bound  to  the 
cell-bodies  themselves.  This  assumption  is  borne  out  by  the  more 
recent  experiments  of  Macfadyen.^  This  author  obtained  acutely 
poisonous  substances  from  pneumococci  by  trituration  of  the  organisms 
after  freezing,  and  extracting  them  with  a  one  1  :  1,000  caustic  potash 
solution.  With  the  filtrates  of  these  extracts  he  was  able  to  cause  rapid 
death  in  rabbits  and  guinea-pigs  by  the  use  of  doses  not  exceeding  0 . 5 
to  1  c.c.  He  found,  furthermore,  a  striking  parallelism  between  the 
degree  of  toxicity  and  the  virulence  of  the  extracted  culture.  Cole,^ 
too,  in  recent  studies,  inclines  to  the  belief  that  the  poisons  of  the  pneu- 
mococcus  are  in  the  nature  of  endotoxins»and  has  produced  toxic  sub- 
stances by  salt  solution  and  bile  extraction  of  the  organisms. 

Immunization. — Recovery  from  a  spontaneous  pneumococcus  in- 

^  G.  and  F.  Klemperer,  Berl.  klin.  Woch.,  xxxiv  and  xxxv,  1891. 

2  Mennes,  Zeit.  f .  Hyg.,  xxv,  1897.  »  p^ne,  Rif.  med.,  xxi,  1898. 

*  Foa  und  Carbone,  Cent.  f.  Bakt.,  x,  1899. 

*  Macfadyen,  Brit.  Med.  Jour.,  ii,  1906. 

6  Cole,  Harvey  Lecture,  N.  Y.,  Dec,  1913, 


DIPLOCOCCUS  PNEUMONIA  363 

f ection  confers  immunity  for  only  a  short  period.  Two  and  three  attacks 
of  lobar  pneumonia  in  the  same  individual  are  not  unusual,  and  it  is 
uncertain  whether  even  a  temporary  immunity  is  acquired  in  such 
infections.  Active  immunization  of  laboratory  animals  may  be  carried 
out  by  various  methods.  The  method  usually  followed  is  to  begin  by 
injecting  attenuated  ^  or  dead  bacteria  or  bacterial  extracts.  Subse- 
quent injections  are  then  made  with  gradually  increasing  doses  of  living, 
virulent  microorganisms.  Great  care  in  increasing  the  dosage  should 
be  exercised  since  the  loss  of  an  animal  after  two  or  three  weeks'  treat- 
ment by  a  carelessly  high  dose  of  pneumococci  is  not  unusual.  Wads- 
worth  centrifugalizes  freshly  grown  pneumococcus  cultures  and  to  the 
pneumococcic  sediment  adds  a  definite  quantity  of  concentrated  salt 
solution.  At  the  end  of  12  hours,  the  pneumococci  are  dead  and  con- 
siderable destruction  of  the  cell-bodies  has  taken  place.  Dilution  with 
water  until  the  solution  equals  0.85  per  cent  NaCl  now  prepares  the 
emulsion  for  inoculation.  The  sera  of  animals  immunized  with  pneu- 
mococci contain  active  bactericidal  substances. 

Specific  agglutinins  in  pneumococcus  immune  sera  were  first 
thoroughly  studied  by  Neufeld  ^  and  since  then  have  been  made  the 
subject  of  extensive  studies  by  Wadsworth,^  Hiss,^  and  many  others. 
For  the  sake  of  obtaining  plentiful  growth  for  agglutination  purposes, 
Hiss  has  recommended  cultivation  in  1%  glucose  broth  with  the  addi- 
tion of  small  amounts  of  sterile  calcium  carbonate  to  absorb  acid  formed 
from  the  glucose.  Pneumococci  do  not  regularly  agglutinate  in  diluted 
immune  sera  and  agglutinations  are  best  studied  in  suspensions  of  more 
concentrated  immune  serum.  Agglutination  begins  at  the  end  of  about 
15  minutes,  and  can  be  studied  both  by  formation  of  clumps  and  by  the 
sediment. 

An  entirely  new  turn  has  been  given  to  studies  of  the  pneumococcus 
group  by  the  observation  by  Neufeld  and  Haendel  that  as  regards  reac- 
tions to  immune  serum  several  varieties  of  pneumococci  exist.  Studies 
based  upon  this  observation  of  Neufeld  ^  and  his  associates  have  more 
recently  been  made  by  Dochez  and  Gillespie.^  These  workers  have 
studied  pneumococci  isolated  from  many  different  human  sources,  both 
by  means  of  protection  in  mice  and  by  agglutination.  They  have  accord- 
ingly divided  pneumococci  into  four  groups,  in  which  they  include,  for 

1  Radziewsky,  Zeit.  f.  Hyg.,  xxxvii,  1901;  Neufeld,  Zeit.  f.  Hyg.,  xi,  1902. 

2  Neufeld,  loc.  cit.         ^  Wadsworth,  loc.  cit.         *  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 
^  Neufeld  and  Haendel,  Arb.  a.  d.  Kais.  Gesund.,  1910,  xxxiv.  293. 

8  Dochez  and  Gillesvie,  Jour.  A.  M.  A.,  1913,  Ixj.  727 


364  PATHOGENIC  MICROORGANISMS 

reasons  mentioned  in  another  place,  the  streptococcus  (seu  pnenmococ- 
cus)  mucosus.  Both  the  methods  of  protection  and  agglutination  per- 
mitted such  a  grouping.  Groups  I  and  II  are  made  up  of  organisms 
that  are  respectively  identical,  since  the  members  of  each  group  react, 
in  protection  and  the  agglutination  experiments,  with  sera  produced 
by  individuals  of  the  same  group.  Group  III  is  distinctive  and  repre- 
sents the  pneumococcus  mucosus,  but  immune  reactions  with  members 
of  this  group  are  very  difficult.  Group  IV  is  a  heterogeneous  collection 
of  organisms  which  have  no  distinctive  characteristics,  and  each  individ- 
ual member  seems  serologically  isolated.  Thus,  the  only  groups  in 
which  exact  serological  experiments  can  be  made  are  those  now  classi- 
fied, as  I  and  11.^ 

In  223  cases  of  lobar  pneumonia  Dochez  finds  the  various  types  dis- 
tributed as  follows: 


Types  of 

Number 

Per 

eumococci 

of  Cases 

Cent. 

I 

78 

34.97 

II 

75 

33.63 

III 

22 

9.86 

IV 

48 

21.52 

Total  number  of  cases         223 

Groups  I,  II  and  III  seem  to  be  most  frequently  associated  with  disr 
ease,  whereas  members  of  Group  IV  are  often  found  in  the  mouths  of 
normal  people.  Groups  I,  II  and  III,  moreover,  are  found  in  the 
sputum  of  normal  individuals  rarely  except  in  association  with  the  dis- 
ease, but  often  in  individuals  who  have  been  closely  associated  with 
cases  of  pneumonia  and  in  convalescents.  Dochez  and  Avery  ^  accord- 
ingly suggest  the  possibility  of  pneumonia  carriers  and  also  come  to  the 
conclusion  that  pneumonia  is  rarely  an  autogenous  infection  of  the  lungs 
with  the  organism  harbored  for  a  long  time  in  the  mouth,  but  is  as  a 
rule  an  infection  with  a  virulent  strain  of  pneumococcus  from  without, 
carriers  furnishing  an  important  element  in  the  transmission  from  pa- 
tients to  the  healthy.  Such  contagiousness  is  also  suggested  by  the 
apparent  epidemics  at  Panama  and  in  Africa,  mentioned  in  a  preceding 
paragraph. 

Precipitins  have  been  demonstrated  in  pneumococcus  immune  serum 

1  Recently  Miss  Olmstead,  in  our  laboratory,  has  obtained  results  which  promise 
the  eventual  subdivision  of  Group  IV  into  definite  divisions. 
^Dochez  and  Avery,  Jour,  of  Exper.  Med.,  1915^  xxi,  114. 


DIPLOCOCCUS  PNEUMONIA  365 

by  Neufeld,  ^  Wadsworth,^  Hiss,^  and  others,  the  organism  for  such  tests 
being  brought  into  solution  either  with  bile  or  with  concentrated  salt 
solution.  Such  sera  also  contain  powerful  opsonic,  substances,  or,  as 
Neufeld  and  Rimpau  ^  prefer  to  call  them,  ''  bacteriotropins. "  It  seems 
most  likely  that  such  phagocytosis-aiding  substances  are  most  power- 
fully concerned  in  protection  and  cure.  Clough  ^  has  reported  an 
increase  of  opsonins  at  the  time  of  crisis,  and  Dochez  ^  has  shown  that 
protective  substances  may  appear  in  the  serum  at  or  soon  after  the 
time  of  crisis.  The  outcome  of  a  case  according  to  Cole  depends 
very  largely  on  the  virulence  of  the  organism  and  on  the  ability  of  the 
body  first  to  limit  the  local  infection  and  to  prevent  the  invasion  of  the 
blood  with  the  organisms.  In  this  process,  of  course,  the  protective 
and  opsonic  bacteriotropic  substances  would  play  a  most  important  part. 

The  history  of  attempts  to  produce  sera  for  passive  immunization 
in  man  is  extensive.  Washburn,^  Mennes,^  Pane^  and  many  others 
in  the  past  have  succeeded  in  protecting  animals  with  such  sera,  but 
with  irregular  results.  The  rational  beginning  based  on  the  recognition 
of  different  pneumococcus  types  was  made  by  Neufeld  and  Haendel  in 
Germany,  and  carried  to  a  considerable  degree  of  success  by  Cole  and 
his  associates  at  the  Rockefeller  Hospital  in  New  York.  By  the  immun- 
ization of  horses  with  the  various  types  of  pneumococci  mentioned 
above,  considerable  success  has  attended  the  use  of  sera  produced 
with  Type  I,  and  less  success  but  great  promise,  that  of  sera  produced 
with  Type  II.  The  injection  of  considerable  quantities  of  the  homolog- 
ous sera  intravenously  at  least  aids  in  sterilizing  the  blood  stream,  and 
upon  this  the  eventual  outcome  of  many  cases  may  depend. 

Since  these  methods  which  at  present  seem  to  promise  the  establish- 
ment of  a  definite  specific  serum  therapy  in  this  serious  disease  depend 
upon  a  rapid  identification  of  the  type  with  which  the  patient  is  infected, 
it  becomes  a  part  of  the  work  of  the  bacteriologist  to  control  the  methods 
which  have  been  developed  for  such  determinations.  The  methods  in 
use  at  the  present  writing  depend  mainly  upon  the  agglutination  of  the 
strains  in  homologous  sera. 

1  Neufeld,  Zeit.  f.  Hyg.,  1902,  xi..  «  Wadsw(yrth,  loc.  cit. 

^  Hiss,  Jour.  Exp.  Med,,  vii,  1905. 

*  Neufeld  and  Rimpau,  Deut.  med.  Woch.,  1904. 

6  Clough,  Johns  Hopkins  Hosp.  Bull.,  Oct.,  1913. 

«  Dochez,  Jour.  Exp.  Med.,  1913. 

'  Washburn,  Brit.  Med.  Jour.,  1897.  «  Mennes,  Zeit.  f.  Hyg.,  1897. 

9  Pane,  Rif .  med.,  1897. 


366 


PATHOGENIC  MICROORGANISMS 


The  patient  is  required  to  wash  out  his  mouth  with  some  antiseptic, 
and  then  expectorate  sputum  obtained  by  coughing,  into  a  Petri  dish. 
The  sputum  is  washed  by  Ufting  it  through  a  series  of  watch  glasses 
containing  sterile  salt  solution  and  is  then  ground  in  a  mortar  with  a 
little  salt  solution,  and  the  even  emulsion  so  obtained  is  injected  intra- 
peritoneally  into  a  white  mouse.  A  fine  needle  is  carefully  introduced 
upward  and  inward  from  just  above  Poupart's  ligament.  Puncture 
of  the  liver  may  be  avoided  in  this  way.  The  pneumococci  develop  in 
the  peritoneum,  and  after  4  to  8  hours  the  mouse  is  killed  and  the  peri- 
toneal cavity  carefully  washed  with  salt  solution  by  means  of  a  capillary 
pipette.  The  washings  are  placed  in  a  centrifuge  tube  at  low  speed  for 
several  minutes  to  bring  down  leucocytes.  The  supernatant  fluid  is 
then  centrifugalized  at  high  speed  and  the  pneumococci  so  obtained  are 
resuspended  in  salt  solution.  This  suspension  is  then  added  to  equal 
parts  of  the  sera  of  Types  I  and  II  in  dilutions  of  1  :  2 . 5  and  1:10,  with, 
of  course,  proper  controls  and  a  bile  test  to  determine  bile  solubility. 
Agglutinations  can  also  be  made  later  with  broth  cultures  made  from 
the  mouse's  heart's  blood  or  peritoneal  exudate. 

Protection  experiments  for  similar  determinations  can  be  carried 
out  by  the  method  shown  in  the  following  protocol  taken  from  the  work 
of  Dochez  and  Avery: 


67  (Group  I) 

A69  (Group  II) 

Dose  of 
Culture 

1 

Con- 

Serum 

Serum 

Con- 

Serum 

Serum 

trols 

I 

II 

trols 

I 

II 

0.1  c.c. 

Dead 

Sur- 

Dead 

Dead 

Dead 

17  hrs. 

vived 

41  hrs. 

18  hrs. 

18  hrs. 

0.01 

17  hrs. 

cc 

25  hrs. 

18  hrs. 

Sur- 
vived 

0.001 

41  hrs. 

tc 

41  hrs. 

18  hrs. 

it 

0.0001 

41  hrs. 

tc 

41  hrs. 

Dead 

18  hrs. 

It 

0.00001 

96  hrs. 

ct 

41  hrs. 

18  hrs. 

u 

0.000001 

48  hrs. 

l( 

72  hrs. 

18  hrs. 

tt 

(The  quantities  in  c.c.  representing  dose  of  culture  refer  to  young 
broth  cultures.) 


DIPLOCOCCUS  PNEUMONIA  367 

No  headway  has  been  made  up  to  the  present  time  with  similar 
treatment  against  Types  III  and  IV. 

Occasional  favorable  results  have  been  obtained  by  Hiss  in  the  treat- 
ment of  pneumococcus  infection  in  animals,  and  by  Hiss  and  Zinsser* 
in  the  treatment  of  pneumonia  in  man  with  aqueous  leucocyte  extracts. 
Although  this  work  has  not  been  extensively  followed  and  has  been  dis- 
appointing in  late  years,  there  is  no  doubt  in  the  mind  of  the  writer 
that  individual  cases  have  been  favorably  influenced.  The  writer  be- 
lieves (differing  from  an  opinion  expressed  in  earlier  papers)  that  the 
influence  of  such  leucocyte  extracts  (as,  it  might  be  incidentally  stated, 
he  believes  the  influence  of  intravenous  injections  of  bacteria  in  typhoid 
and  other  conditions)  consists  in  its  chemotactic  influence,  i.e.,  its  ten- 
dency to  increase  circulating  leucocytes.  The  contrast  between  the  good 
results  in  rabbits  and  the  less  striking  results  in  man  may  easily  be  due 
to  the  fact  that  in  rabbits  intravenous  injections  were  practiced. 

Differentiation  of  Pneumococcus  from  Streptococcus. — Pneumococci 
and  streptococci  which  do  not  differ  in  morphology  from  their  classic 
types  can  usually  be  differentiated  from  each  other  and  identified  by 
their  morphological  characters  without  difficulty;  but 'it  is  equally  true 
that  certain  cultures  of  these  organisms,  either  at  the  time  of  their 
isolation  or  after  cultivation  on  artificial  media,  approach  the  type  of  the 
other  so  closely  that  it  may  be  impossible  to  identify  them  by  their  mor- 
phology alone.  When  such  morphological  variations  occur  there  are  no 
constant  cultural  or  pathogenic  characters  as  yet  demonstrated  which 
distinguish  between  these  organisms. 

This  lack  of  distinct  cultural  differences  between  pneumococci  and 
streptococci  has  not  infrequently  led  to  confusion,  and  that  uncertainty 
should  exist  and  mistakes  be  made  in  identification  is  not  surprising 
when  one  considers  the  characters  usually  depended  upon  to  distinguish 
pneumococci  from  streptococci.  Chief  among  these,  as  has  just  been 
implied,  are  the  morphological  features  which  are,  in  the  case  of  pneu- 
mococci, a  slightly  lancet  or  elongated  form  rather  than  the  more  typical 
coccus  form  characteristic  of  the  streptococci,  and  an  arrangement  of 
such  cocci  in  pairs  rather  than  in  chains;  added  to  these  features  is  the 
possession  of  a  more  or  less  well-defined  capsule.  All  of  these  char- 
acters are  subject  to  variation  or  may  be  absent.  Compared  with  the 
morphological,  the  cultural  characters  are  variable  and  of  minor  im- 
portance.    The  pneumococcus  colonies  on  coagulated  blood  serum  and 

*  Hiss  and  Zinsser,  Jour.  Med.  Res.,  xix,  1908. 


368  PATHOGENIC  MICROORGANISMS 

on  agar  are  moister  and  flatter,  and  the  freshly  isolated  pneumococcus 
is  usually  unable  to  develop  readily  or  at  all  on  gelatin  at  below  22°  C. 
The  distinctness  of  the  capsule  of  the  pneumococcus  in  the  body 
fluids  of  man  and  animals  and  on  blood  serum,  milk,  or  serum  agar, 
has  been  depended  upon  as  the  chief  distinguishing  and  diagnostic 
character.  Nevertheless,  instances  have  been  reported  of  distinct  cap- 
sule formation  by  organisms  which  had  either  been  previously  iden- 
tified as  Streptococcus  pyogenes,  or  at  the  time  of  their  isolatoin  could 
not  be  definitely  identified  as  belonging  to  this  group  or  to  the  pneu- 
mococci,  but  were  considered  intermediate  in  character.^ 

1  Brief  Description  of  Organisms  Reported  as  Capsulated  Streptococci. — Bordet 
{Bordet,  Ann.  de  I'inst.  Pasteur,  1897,  xi,  p.  177),  working  with  an  organism  previously- 
identified  as  Streptococcus  pyogenes,  described  such  capsule  formation  occurring  in 
the  peritoneal  exudate  of  infected  rabbits. 

Schuetz'  (Schuetz,  Cent.  f.  Bakt.,  Ref.  1,  1887,  p.  393)  Diplokokkus  der  Brust- 
seuche  der  Pferde,  Poels  and  Nolen's  {Poels  und  Nolen,  Fort.  d.  Med.,  iv,  1886,  p.  217) 
streptococcus  of  contagious  pneumonia  of  cattle,  and  especially  the  organism  de- 
scribed by  Bonome  (Bonome,  Ziegler's  Beit.,  viii,  1890,  p.  377)  as  Streptococcus 
der  meningitis  cerebrospinalis  epidemica,  may  all  be  looked  upon  as  organisms 
differentiated  on  insecure  grounds  from  either  pneumococcus  or  streptococcus.  The 
first  two  of  these  organisms,  however,  are  said  to  be  decolorized  by  Gram's  method, 
and  as  suggested  by  Frosch  and  KoUe  {Frosch  und  Kolle,  Fliigge's  "  Mikro-organis.," 
ii,  1896,  p.  161),  in  the  case  of  Schuetz'  organism  may  belong  to  a  group  inter- 
mediate between  Fraenkel's  diplococcus  and  the  chicken-cholera  group. 

Tavel  and  Krumbein  (Tavel  und  Krumhein,  Cent.  f.  Bakt.,  xviii,  1895,  p.  547) 
describe  a  streptococcus  with  a  capsule,  which  was  isolated  from  a  small  abscess  on 
the  finger  of  a  child.  Capsules  were  also  present  in  the  artificial  cultures,  and  al- 
though ordinarily  remaining  uncolored,  could  be  stained  by  Loeffler's  flagella  stain. 
This  organism  was  said  to  be  differentiated  from  Fraenkel's  diplococcus  and  also  in 
general  from  streptococcus  (pyogenes)  by  a  rapid  and  rich  growth  on  gelatin,  agar, 
and  potato.  A  pellicle  was  formed  on  broth.  The  organisms  forming  this  pellicle 
had  capsules,  but  those  in  the  deeper  portions  of  the  broth  generally  lacked  it. 

In  1897,  Binaghi  (Binaghi,  Cent.  f.  Bakt.,  xxii,  1897,  p.  273)  described  a  cap- 
sulated streptococcus  isolated  from  a  guinea-pig  dead  of  a  spontaneous  peribron- 
chitis and  multiple  pulmonary  abscesses.  In  the  pus  were  found  some  diplococci  and 
short  chains  (four  to  six)  surrounded  by  a  capsule,  shown  by  staining  with  carbol 
fuchsin.     This  organism  he  proposes  to  call  Streptococcus  capsulatus. 

Le  Roy  des  Barres  and  Weinberg  in  1899  (Le  Roy  des  Barres  et  Weinberg,  Arch, 
d.  m6d.  exper.,  xi,  1899,  p.  399)  published  an  account  of  a  streptococcus  with  a 
capsule.  This  was  isolated  from  a  man  who  had  apparently  been  infected  from  a 
horse  which  had  died  of  an  acute  intestinal  disorder.  The  patient  neglected  the 
infection  and  died.  Diplococci  and  short  chains  furnished  with  a  capsule  were 
found  in  the  subcutaneous  tissue  at  the  area  of  infection.  The  blood,  hver,  and 
spleen  also  contained  these  organisms.  The  capsule  in  all  the  preparations  remained 
uncolored,  but  the  authors  say  that  its  existence  was  not  to  be  doubted.    Ascitic 


DIPLOCOCCUS  PNEUMONIiE  369 

There  are  occasions,  then,  both  within  the  animal  body  and  in  arti- 
ficial cultivations,  when  it  is  practically  impossible  to  distinguish  defi- 
nitely between  some  races  of  pneumococci  and  races  of  streptococci. 
This  difficulty  is  especially  heightened  when  the  pneumococcus  has 
become  non-virulent,  and  at  the  same  time  no  very  typical  morphology 
or  capsule  formation  is  to  be  determined  and  a  tendency  to  chain-forma- 
tion is  marked.  Cultures  of  pneumococci  in  such  condition  can  not 
readily  be  distinguished  morphologically  from  streptococcus  cultures. 

Under  these  circumstances  recourse  must  be  had  to  a  careful  bio- 
logical study  of  the  organism  in  question.  The  following  are  the  criteria 
mainly  relied  upon  at  present  for  the  differentiation  of  these  two  groups. 

Pneumococci  ferment  inulin,  if  cultivated  in  inulin-serum-water  me- 
dium. Acid  formation  from  the  inulin  results  within  two  days  or  more 
in  coagulation  of  the  serum  and  reddening  of  the  litmus.  Streptococci  be- 
cause of  their  inability  to  attack  the  inulin  leave  the  mediimi  unchanged.' 

broth  inoculated  from  the  peritoneal  exudate  of  a  rabbit  dying  from  the  infection 
gave  streptococci  in  extremely  long  chains  and  surrounded  by  capsules.  These  were 
not  so  distinct  as  in  the  case  of  the  organisms  in  the  original  smear  preparations. 
All  fluid  media  (bouillon,  milk,  and  ascitic  broth)  were  said  to  be  strongly  acid  after 
twenty-four  hours.  These  authors  report  that  Achard  and  Marmorek  have  assured 
them  that  they  have  seen  capsulated  streptococci,  and  that  Marmorek  showed  them 
some  preparations  in  which  one  of  his  streptococci  presented  the  same  characters  as 
that  isolated  by  them. 

Although  Le  Roy  des  Barres  and  Weinberg  have  used  the  term  encapsulated, 
they  beheve  that  it  would  perhaps  be  more  prudent  to  call  their  organism  strepto- 
coque  aureole,  since  they  were  not  able  to  define  this  capsule  by  staining  it. 

Howard  and  Perkins  {Howard  and  Perkins,  Jour.  Med.  Res.,  1901,  iv,  p.  163) 
have  lately  described  an  organism,  probably  of  the  foregoing  type,  which  was  present 
in  a  tubo-ovarian  abscess  and  in  the  peritoneal  exudate,  the  blood,  and  some  of  the 
organs  of  a  woman  dying  in  the  Lakeside  Hospital,  Cleveland,  Ohio.  The  organisms 
were  biscuit-shaped  cocci  in  pairs,  usually  arranged  in  chains  of  four,  six,  eight, 
or  twenty  elements,  and  surrounded  by  a  wide  and  sharply  staining  capsule.  In  the 
artificial  cultures  special  capsule  stains,  it  was  noted,  failed  to  stain  any  definite  area, 
but  numerous  small  deeply  stained  jsranules  were  to  be  seen  within  the  halo,  espe- 
cially near  its  outer  border.  Howard  and  Perkins  propose  for  the  group  composed  of 
the  streptococci  of  Bonome,  Binaghi,  and  their  own  organism,  the  name  Strepto- 
coccus mucosus.  Streptococci  isolated  from  cases  of  epidemic  sore-throat  have  also 
shown  capsules  (p.  343). 

Reference  to  the  original  descriptions  of  these  various  capsulated  streptococci 
will  show  that,  with  the  exception  of  a  rather  poorly  staining  capsule,  the  majority  of 
these  organisms  are  separated  from  the  typical  Streptococcus  pyogenes  or  from  the 
pneumococcus  by  exceedingly  slight  and  unstable  morphological  and  cultural  char- 
acters.    This  is  true  of  the  difference  in  their  pathogenic  action  in  animals. 

1  Hiss,  Cent.  f.  Bakt.,  xxxi,  1902;  Jour.  Exp.  Med.,  vi,  1905. 


370  PATHOGENIC  MICROORGANISMS 

Cultivated  on  whole-blood-agar,  streptococci  usually  cause  hemo- 
lysis, pneumococci  usually  do  not.^  In  contradistinction  to  Streptococ- 
cus viridans  which  does  not  hemolyze,  pneumococci  have  a  tendency  on 
these  media  to  form  the  black,  dry,  paint-blister  colonies.^ 

Neufeld,^  in  1900,  noticed  that  normal  rabbits'  bile  added  in  quan- 
tities of  0.1  c.c.  to  each  one  or  two  cubic  centimeters,  of  a  pneumococcus 
broth  culture  caused  lysis  of  the  bacteria,  rendering  the  culture  fluid 
transparent  and  clear.  This  does  not  occur  with  streptococci,  and  has 
been  used  to  differentiate  the  two  species.  According  to  Libman  and 
Rosenthal,^  great  reliance  may  be  placed  upon  this  method. 

The  most  convenient  reagent  for  use  in  the  Neufeld  bile  test  is  a 
10  per  cent  solution  of  sodium  taurocholate  in  physiological  salt  solution. 
This  should  be  sterilized  or  kept  on  ice.  One-tenth  volume  of  such  a 
solution  produces  prompt  lysis  in  a  broth  culture  of  pneumococci. 

Decisive  differential  importance  may  be  attached  to  the  agglutina- 
tions of  these  microorganisms  in  immune  sera  (see  p.  364). 

The  permanency  of  the  various  types  in  the  pnemnococcus-strepto- 
coccus  group  is  still  open  to  question.  E.  C.  Rosenow  ^  has  recently  re- 
ported that  he  has  transmuted  typical  pneumococci  into  typical  hemo- 
lytic streptococci  by  methods  which  he  has  not  as  yet  fully  described,  but 
among  which  were  animal  passage,  growth  in  symbiosis  with  bacillus 
subtilis,  and  growth  in  an  atmosphere  of  oxygen.  The  pneumococci 
when  first  altered  took  on  the  characteristics  of  the  streptococcus 
viridans,  later  of  the  so-called  streptococcus  rheumaticus,  and  finally 
of  streptococcus  hemolyticus.  Together  with  cultural  characteristics 
the  pathogenicity  of  these  various  strains  for  rabbits  changed.  The 
pneumococcus  produced  acute  sepsis,  the  streptococcus  viridans  caused 
endocarditis,  the  streptococcus  'rheumaticus  periarticular  or  serous 
arthritis,  and  hemolyticus  suppurative  arthritis.  In  intermediate  stage 
the  organisms  quite  regularly  caused  myositis.  Although  he  was  able 
to  transmute  these  types  one  into  the  other  in  both  directions,  Rose- 
now believes  that  the  cultural  characteristics  of  each  type  correspond 
to  a  fairly  definite  type  of  pathogenicity  in  animals  and  man.  This 
work  has  not  as  yet  appeared  in  detail  and  has  not  been  confirmed. 

*  Schottmiiller,  Miinch.  med.  Woch. 
2  Hiss,  Jour.  Exp.  Med.,  vii,  1905. 

*  Neufeld,  Zeit.  f.  Hyg.,  1901. 

*  Libman  and  Rosenthal,  Proc.  N.  Y.  Path.  Soc,  March,  1908. 

*  Rosenow,  J.  A.  M.  A.,  1913,  Ixi,  2007, 


CHAPTER  XXIV 

MICROCOCCUS   INTRACELLULARIS   MENINGITIDIS 
(MENINGOCOCCUS) 

Infectious  processes  in  the  meninges  may  be  caused  by  many  dif- 
ferent microorganisms. 

Meningitis  may  be  primary  or  secondary.  Secondary  meningitis 
may  often  occur  during  the  course  of  pneumonia,  when  pneumococci, 
carried  to  the  meninges  by  the  blood  stream,  give  rise  to  a  usually  fatal 
form  of  the  disease.  More  rarely  a  similar  process  may  occur  as  a 
secondary  manifestation  of  typhoid  fever  or  influenza.  Meningitis  may 
also  result  secondarily  by  direct  extension  from  suppurative  lesions  about 
the  skull,  such  as  those  occurring  in  diseases  of  the  middle  ear  or  frontal 
sinuses  or  after  compound  fractures.  In  such  cases  the  invading  or- 
ganisms are  usually  staphylococci,  streptococci,  or  pneumococci. 

Isolated  cases  of  meningeal  infection  with  B.  coli,  B.  paratyphosus. 
Bacillus  pestis,  and  Bacillus  mallei  have  been  reported.  A  frequent, 
more  chronic  form  of  the  disease  is  caused  by  Bacillus  tuberculosis. 

Primary  acute  meningeal  infection,  however,  is  due  chiefly  to  two 
microorganisms,  Micrococcus  intracellularis  meningitidis,  and  the  pneu- 
mococcus. 

A  tabulation  of  the  comparative  frequency  with  which  the  various 
microorganisms  are  found  in  the  meninges  has  been  attempted  by 
Marschal.^  This  author  estimates  that  about  69.2  per  cent  of  all 
acute  cases  are  due  to  the  meningococcus,  20.8  per  cent  to  Diplococcus 
pneumoniae,  and  the  remaining  10  per  cent  to  the  other  bacteria 
mentioned. 

The  cases  caused  by  the  pneumococcus  and  the  other  less  frequent 
incitants  usually  occur  sporadically.  When  the  disease  occurs  in  epi- 
demic form,  it  is  almost  always  due  to  the  meningococcus. 

Diplococcus  intracellularis  meningitidis  was  first  seen  in  menin- 
geal exudates  by  Marchiafava  and  Celli  ^  in  1884.  These  authors  not 
only  described  accurately  the  morphological  characteristics  now  recog- 

1  Marschal,  Diss.  Strassburg,  1901,  Quoted  from  Weichselbaum,  in  Kolle  und 
Wassermann,  "  Handbuch." 

2  Marchiafava  and  Celli,  Gaz.  degli  ospedali,  8,  1884. 

871 


372  PATHOGENIC   MICROORGANISMS 

nized,  but  also  called  attention  to  the  intracellular  position  of  the  micro- 
organism and  to  its  gonococcus-like  appearance.  They  failed,  however, 
to  cultivate  it. 

Observations  confirmatory  of  the  Italian  authors  were,  soon  after, 
made  by  Leichtenstern.^  Cultivation  and  positive  identification  as  a 
separate  species  was  not  accomplished,  however,  until  Weichselbaum,^ 
in  1887,  reported  his  observations  upon  six  cases  of  epidemic  cerebro- 


'  y^  ^  \, 

".     .V    ^^ 

• 

# 

^^         e 

#    # 

• '    '^'  ^  . 

•»       »•••'• 

• 
• 

%                      % 

"   It    *     ♦* 

♦                 % 

.V.    •. 

W- 

'  .li  ■ 

Fig.  77. — Meningococcus,  Pure  Culture.    (Very  highly  magnified.) 

spinal  meningitis  in  which  he  not  only  found  the  cocci  morphologically, 
but  was  able  to  study  their  biological  characteristics  in  pure  culture. 
The  researches  of  Weichselbaum  were  soon  confirmed  and  extended 
by  elaborate  studies  ^  which  left  no  doubt  as  to  the  specific  relationship 
between  the  microorganism  cultivated  by  him  and  the  clinical  condition. 

1  Leichtenstern,  Deut.  med.  Woch,,  1885. 

2  Weichselbaum,  Fort.  d.  Med.,  1887. 

^Councilman,  Mallory,  and  TFngrA^,  Special  Rep.  Mass.  Board  of  Health,  1898; 
AUbrecht  und  Ghon,  Wien.  klin.  Woch.,  1901. 


MICROCOCCUS  INTRACELLULARIS  MENINGITIDIS 


373 


Morphology  and  Staining. — Stained  in  the  spinal  fluid  from  an  in- 
fected patient,  the  meningococcus  bears  a  striking  similarity  to  the  gon- 
ococcus.  The  microorganisms  appear  intra-  and  extracellularly,  usually 
in  diplococcus  groups,  sometimes  as  tetrads,  or  even  in  larger  agglomer- 
ations. The  individual  diplo-forms  are  flattened  on  the  sides  facing  each 
other,  presenting  somewhat  the  biscuit-form  of  the  gonococcus.  The 
variation  in  size  of  the  cocci  in  the  same  smear  is  a  noticeable  feature 


Fig.  78, — Meningococcus  in  Spinal  Fluid.  - 

and  of  some  diagnostic  importance.  This  dissimilarity  in  size  is  notice- 
able also  in  cultures,  which,  especially  when  older  than  twenty-four 
hours,  contain  forms  double  or  even  triple  the  size  of  the  average  coccus. 
These  may  possibly  be  involution  forms. 

The  meningococcus  is  non-motile  and  non-spore  forming.  It 
stains  easily  with  all  the  usual  aqueous  anilin  dyes.  Its  behavior 
toward  Gram's  stain  was  long  a  subject  of  controversy,  owing  to  the 
error  of  Jaeger,^  who  claimed  to  have  found  it  Gram-positive.    There 

^  Jaeger,  Zeit.  f.  Hyg.,  xix,  1895. 


374  PATHOGENIC  MICROORGANISMS 

is  no  question  now,  however,  that  the  cocci  decolorize  by  Gram's  method 
when  this  is  carefully  carried  out. 

In  spinal  fluid  very  beautiful  preparations  may  be  obtained 
by  staining  in  Jenner's  blood  stain.  Councilman,  Mallory,  and 
Wright  ^  were  the  first  to  notice  that,  when  stained  with  Loeffler's 
methylene-blue,  meningococcus  stains  irregularly,  showing  metachro- 
matic granules  in  the  center  of  the  cell  bodies.  These  granules  can  be 
demonstrated  more  clearly  with  the  Neisser  stain  employed  for  similar 
demonstration  in  the  case  of  B.  diphtherise  (see  p.  107)  and  have  some 
value  in  differentiating  meningococcus  from  gonococcus. 

Cultivation. — Micrococcus  intracellularis  meningitidis  grows  readily 
upon  all  the  meat-infusion  culture  media.  It  may  even  be  culti- 
vated upon  meat-extract  media,  but  growth  upon  these  is  not  profuse. 
Upon  agar,  colonies  appear  within  eighteen  to  twenty-four  hours  as 
grayish,  glistening  spots  with  smooth  edges  and  raised  granular  pentere. 
These  show  a  tendency  to  enlargement  and  eventual  confluence. 

Growth  is  more  luxuriant  and  rapid  upon  media  to  which  animal 
proteid  in  the  form  of  blood  serum  or  ascitic  fluid  has  been  added.  Co- 
agulated serum  is  not  liquefied.  For  cultivation  of  the  meningococcus 
directly  from  the  human  body  it  is  wise  to  use  the  richer  serum  or  blood 
media,  ability  to  grow  easily  upon  simple  agar  being  occasionally  acquired 
only  after  previous  cultivation  upon  richer  media.  Agar  to  which  whole 
rabbit's  blood  has  been  added  forms  an  excellent  medium,  both  for  cul- 
tivation and  for  keeping  the  organism  alive.  Loefjiefs  blood  serum 
is  also  very  favorable.  It  is  advisable,  too,  when  cultivating  directly 
from  spinal  fluid,  to  plant  rather  large  quantities  (1  to  2  c.c),  since 
many  of  the  cocci  in  the  exudate  will  fail  to  develop  colonies,  possibly 
because  of  their  prolonged  exposure  either  to  the  body  fluids  or  to  their 
own  products  in  a  closed  space. 

Upon  broth,  growth  is  slow  and  takes  place  chiefly  upon  the  surface, 
the  sediment  consisting  mainly  of  dead  bacteria.  Glucose  added  to  agar 
or  to  broth  renders  the  medium  more  favorable  for  rapid  growth,  but, 
owing  to  acid  formation,  tends  to  cause  a  more  rapid  death  of  the  culture. 
In  flasks  of  broth  containing  glucose  one  per  cent,  and  CaCOg  one  per- 
cent, however,  cultures  have  been  kept  alive  for  as  long  as  fourteen 
months  (Hiss).  On  milk,  growth  takes  place  without  coagulation 
of  the  casein.  Potatoes  are  not  a  favorable  medium,  though  growth 
occasionally  takes  place. 

^  Councilman,  Mallory,  and  Wright,  Rep.  Mass.  State  Bd.  of  Health,  1898. 


MICROCOCCUS    INTRACELLULARIS  MENINGITIDIS 


375 


While  slight  alkahnity  or  acidity  does  not  inhibit,  the  most  favor- 
able reaction  of  media  is  neutrality. 

Oxygen  is  necessary  for  development.  Complete  anaerobiosis,  while 
not  absolutely  inhibitory,  is  extremely  unfavorable,  unless  proper 
carbohydrates  be  present. 

While  growth  may  take  place  at  temperatures  ranging  from  25° 


1  /,'■.  ^i^.^ 

'I' 

1 ^ 1 

Fig.  79. — ^Meningococcus  Culture.      Streak   culture   from  spinal  fluid  on 

serum  agar-plate. 

to  42°  C,  the  optimum  is  37.5°  C.  Apart  from  the  remarkable  viability 
displayed  upon  calcium-carbonate  broth,  the  average  length  of  time 
during  which  the  meningococcus  will  remain  alive  without  transplanta- 
tion is  rather  short.  Recently  isolated  cultures  grown  on  agar  or  serum- 
agar  may  die  within  two  or  three  days.  Accustomed  to  artificial  cul- 
tivation throu-gh  a  number  of  generations,  however,  the  cultures  become 


376  PATHOGENIC  MICROORGANISMS 

more  hardy  and  transplantation  may  safely  be  delayed  for  a  week  or 
even  longer.  Albrecht  and  Ghon  ^  have  kept  a  culture  alive  on  agar 
for  one  hundred  and  eighty-five  days.  It  is  a  strange  fact  that  after 
prolonged  artificial  cultivation  some  strains  of  meningococcus  may 
gradually  lose  their  growth  energy  and  finally  be  lost  because  of  their 
refusal  to  develop  in  fresh  transplants.  Storage  is  best  carried  out  at 
incubator  temperatures.  At  room  temperatures  or  in  the  ice  chest, 
the  diplococcus  dies  rapidly .^ 

Resistance. — ^The  meningococcus  is  killed  by  exposure  to  sunlight  or 
to  drying  within  twenty-four  hours. ^  It  is  extremely  sensitive  to  heat 
and  cold  and  by  the  common  disinfectants  is  killed  in. high  dilutions 
and  by  short  exposures.  At  0°  C.  it  usually  dies  within  two  or  three 
days. 

Pathogenicity. — As  stated  above,  the  form  of  meningitis  caused  by 
the  diplococcus  of  Weichselbaum  occurs  usually  in  epidemics,  though 
isolated  sporadic  cases  are  seen  from  time  to  time  in  all  crowded  com- 
munities. Epidemics  have  been  numerous  and  widespread,  and  their 
records  far  antedate  the  discovery  of  their  causative  agent.  As  a  rule, 
these  epidemics  have  occurred  during  the  winter  and  spring  months, 
and  have  attacked  chiefly  that  part  of  the  population  which  is  forced  by 
poverty  to  live  in  crowded  unhygienic  surroundings.  The  manner  in 
which  the  microorganism  enters  the  human  body  is  still  a  subject  for 
investigation.  Weichselbaum,*  Ghon  and  Pfeiffer,^  and,  more  recently, 
Goodwin  and  v.  Sholly  ®  of  the  New  York  Department  of  Health,  have 
succeeded  in  demonstrating  culturally  the  presence  of  the  meningococ- 
cus in  the  nasal  cavities,  not  only  of  patients  suffering  from  the  disease, 
but  occasionally  in  those  of  healthy  subjects  as  well.  Similar  findings 
have  been  reported  by  many  others;  but  in  many  cases  morphological 
examination  only  was  made,  which,  owing  to  the  danger  of  confusion 
with  Micrococcus  catarrhalis,  a  frequent  inhabitant  of  the  nose,  renders 
such  reports  valueless.  The  careful  work  of  the  writers  mentioned,  how- 
ever, has  given  ground  for  the  theory  that  meningeal  infection,  which  is 

1  Albi^echt  und  Ghon,  Wien.  klin.  Woch.,  1901. 

2  A  very  thorough  biological  study  of  meningococcus  and  related  organisms  has 
recently  been  made  by  Elser  and  Huntoon  (Jour.  Med.  Res.,  N.  S.  vol.  xv,  1999), 
which  may  be  consulted  for  a  more  detailed  description  of  cultural  characteristics. 

3  Councilman,  Mallory,  and  Wright,  Boston,  1898;  Albrecht  and  Ghon,  loc.  cit. 
*  Weichselbaum,  Fort.  d.  Med.,  1887. 

^Ghon  und  Pfeiffer,  Zeit.  f.  klin.  Med.,  xliv,  1901. 

« Goodwin  und  v.  Sholly,  Jour.  Inf.  Dis.,  Suppl.  2,  Feb.,  1906. 


MICROCOCCUS  INTRACELLULARIS  MENINGITIDIS  377 

often  preceded  by  nasal  catarrh,  may  take  place  along  the  paths  of  the 
lymphatics,  passing  out  of  the  nose  and  its  accessory  cavities  toward  the 
base  of  the  skull.  These  facts,  together  with  the  low  resistance  shown 
by  the  meningococcus  against  drying,  and  the  general  failure  so  far  to 
demonstrate  it  in  air,  dust,  or  fomites,  would  seem  to  indicate  that  trans- 
mission usually  occurs  directly  from  one  human  being  to  another. 

The  disease  produced  in  man  consists  anatomically  in  a  suppurative 
lesion  of  the  meninges,  involving  the  base  and  cortex  of  the  brain  and  the 
surface  of  the  spinal  cord.  The  nature  of  the  exudate  may  vary  from  a 
slightly  turbid  serous  fluid  to  that  of  a  thick  fibrinous  exudate.  In 
chronic  cases  encephalitis  and  dilatation  of  the  ventricles  may  take 
place.  Apart  from  their  presence  in  the  meninges  and  in  the  naso- 
pharynx, meningococci  have  not  been  satisfactorily  demonstrated  in 
any  of  the  complicating  lesions  of  the  disease.  Reports  of  their  presence 
in  the  conjunctivae,  in  the  bronchial  secretions  from  broncho-  or  lobar 
pneumonia,  and  in  otitis  media,  have  usually  been  based  upon  insuf- 
ficient bacteriological  evidence. 

The  occurrence  of  this  microorganism  in  the  circulating  blood  of  men- 
ingitis cases  has  been  definitely  proved  by  Elser,^  who  found  it  in  ten 
cases. 

Animals  are  not  very  susceptible  to  infection  with  Diplococcus 
meningitidis.  Subcutaneous  inoculation  is  rarely  followed  by  more 
than  a  local  reaction  unless  large  quantities  are  used.  White  mice  are 
rather  more  susceptible  than  other  species.  Intraperitoneal  and  intra- 
venous inoculation  of  sufficient  quantities  usually  results  in  the  death 
of  mice,  rabbits,  guinea-pigs,  and  dogs.  Occasional  strains  have  been 
found  to  possess  a  not  inconsiderable  degree  of  toxicity  for  rabbits, 
grave  symptoms  or  even  death  following  intravenous  injection  of  but 
moderate  quantities  without  any  traceable  development  of  the  micro- 
organisms in  the  organs  of  the  animals. 

Similar  observations  have  been  made  by  Albrecht  and  Ghon,^  who 
succeeded  in  killing  white  mice  with  dead  cultures.  It  would  seem 
therefore  that  the  effect  of  this  coccus  upon  animals  depends  chiefly 
upon  the  poisonous  substances  contained  in  the  bacterial  bodies  (endo- 
toxins) .  Lepierre  ^  has  obtained  the  meningococcus  toxin  by  alcohol 
precipitation  of  broth  cultures. 

Weichselbaum  himself  succeeded  in  producing  meningeal  suppura- 

1  Elser,  Jour.  Med.  Res.,  xiv,  1906. 

2  Albrecht  und  Ghon.,  loc.  cit. 

*  Lepierre,  Jour,  de  phys.  et  de  path.  g6n.,  v,  No.  3. 
25 


378  PATHOGENIC  MICROORGANISMS 

tion  and,  in  one  case,  brain  abscess,  by  subdural  inoculation  of  dogs. 
Councilman,  Mallory,  and  Wright  ^  produced  a  disease  in  many  re- 
spects similar  to  the  human  disease  by  intraspinous  inoculation  of  a 
goat.  Recently,  Flexner^  has  succeeded  in  producing  in  monkeys  a 
condition  entirely  analogous  to  that  occurring  in  human  beings. 

Agglutination. — Immunization  of  animals  by  repeated  inoculations 
of  meningococcus  ^  results  in  the  formation  in  the  blood  serum  of 
agglutinins.  KoUe  and  Wassermann  *  obtained  from  horses  a  serum 
which  had  an  agglutinating  value  of  I  :  3,000  for  the  homologous 
strain,  and  of  as  much  as  1  :  500  for  other  true  meningococcus 
strains.  Similar  experiments  by  Dunham  ^  and  others  have  proved  the 
unquestionable  value  of  agglutination  for  species  identification  of  this 
group.  Great  differences  m^y,  however,  exist  between  individual 
races  in  their  aggiutinability  in  the  same  immune  serum. 

Kutscher  has  recently  called  attention  to  the  fact  that  strains 
which  can  not  be  agglutinated  in  specific  sera  at  37°  C.  will  often  yield 
positive  results  when  subjected  to  55°  C,  a  fact  of  some  practical  im- 
portance if  confirmed. 

Elser  and  Huntoon  ^  have  shown  that  in  the  serum  of  infected  human 
subjects  agglutination  of  some  strains  takes  place  in  dilutions  as  high 
as  1  :  400. 

Serum  Therapy  of  Meningitis. — During  recent  years,  attempts  have 
been  made  to  treat  epidemic  meningitis  by  injections,  subcutaneous 
and  intraspinous,  of  meningococcus-immune  serum.  Wassermann,^  in 
1907,  reported  results  of  such  treatment  in  one  hundred  and  two  patients, 
with  a  recovery  of  32 . 7  per  cent.  The  serum,  manufactured  by  Was- 
sermann and  his  associates,  was  obtained  from  horses  immunized  with 
cultures  of  meningococcus  and  with  toxic  meningococcus  extracts. 
More  recently  Flexner  and  Jobling  ^  have  used  a  similar  serum  in 
the  United  States  with  apparently  excellent  results.  The  serum,  in 
Flexner's  cases,  as  in  the  technique  of  Jochmann,  is  injected 
intraspinously  after  a  quantity  of  spinal  fluid  had  been  withdrawn. 
The  cases  treated  by  Flexner  and  Jobling' s  method  have  now  reached 

1  Councilman,  Mallory,  and  Wright,  loc.  cit. 

2  Flexner,  Jour.  Exp.  Med.,  1906. 

3  Albrecht  and  Ghon,  Wien.  klin.  Woch.,  1901. 

■  Kolle  und  Wassermann,  Deut.  med.  Woch.,  15,  1906. 

*  Dunham,  Jour.  Inf.  Dis.,  11,  1907. 

«  Elser  and  Huntoon,  loc.  cit. 

»  Wassermann,  Deut.  med.  Woch.,  39,  1907.    . 

«  Flexner  and  Joblinq,  Jour.  Exper.  Med.,  x,  1908. 


MICROCOCCUS  INTRACELLULARIS  MENINGITIDIS  379 

large  numbers,  both  in  this  and  foreign  countries  and  the  value  of  the 
serum  as  a  therapeutic  agent  seems  firmly  established. 

Hiss  and  Zinsser  ^  have  treated  a  number  of  meningitis  patients  with 
subcutaneous  injections  of  leucocyte  extracts  and  believe  that  they  have 
favorably  influenced  the  course  of  the  disease. 

Pseudomeningococcus. — Elser  and  Huntoon  ^  have  described  a  diplo- 
coccus  very  similar  to  the  meningococcus  which  they  differentiated 
from  it  only  by  serum  reactions.  This  diplococcus  could  be  identified 
only  by  agglutinin  absorption  tests.  They  named  it  pseudomeningo- 
coccus. 

Parameningococcus. — Dopter^  described  a  Gram-negative  diplococ- 
cus which  was  identical  with  meningococcus  in  its  cultural  and  fer- 
mentative characteristics.  It  failed  to  give  agglutination  and  pre- 
cipitation reactions  in  anti-meningitis  serum,  though  it  did  give  a 
complement-fixation  reaction.  These  organisms,  at  first  isolated  from 
normal  throats,  have  since  been  found  in  the  spinal  fluid  of  cases  of 
meningitis.  In  a  recent  study  of  strains  from  meningitis  Amoss  and 
Wollstein  found  that  besides  the  two  distinct  types — normal  menin- 
gococci and  parameningococci — there  were  a  number  of  intermediate 
varieties.  Olmstead  and  Dubois*  and  Neal  and  Schweitzer^  also  found 
marked  antigenic  differences  among  the  strains  of  meningococci  which 
they  studied.  The  use  of  representatives  of  each  of  the  types  of 
meningococci  and  parameningococci  has  been  found  essential  for  the 
production  of  a  potent  anti-meningitis  serum. 

1  Hiss  and  Zinsser,  Jour.  Med.  Res.,  Nov.,  1908. 

2  Elser  and  Huntoon,  Jour.  Med.  Res.,  xxi,  1909. 

3  Dopter,  "Conte  Rendu  de  la  Society  de  Biologie,"  1909,  Ixvii,  p.  74, 
*  Olmstead  and  Dubois,  Jour.  Exper.  Med.,  xxiii,  p.  403. 

5  Neal  and  Schweitzer,  Jour,  of  Immunol.,  1916,  i,  p.  307* 


CHAPTER  XXV 

DIPLOCOCCUS     GONORRHCEiE     (GONOCOCCUS),    MICROCOCCUS 
CATARRHALIS,    AND    OTHER     GRAM-NEGATIVE    COCCI 

DIPLOCOCCUS   GONORRHCEiE  4 

Neisser/  in  1879,  described  diplococci  which  he  had  found  regulai^^ 
in  the  purulent  secretions  of  acute  cases  of  urethritis  and  vaginitis  arfd 
in  the  acute  conjunctivitis  of  the  new-born.  His  researches  were  purely 
morphological,  as  were  the  numerous  confirmatory  investigations  which 
rapidly  followed  his  announcement. 

Cultivation  of  this  diplococcus,  now  usually  spoken  of  as  gonococcus, 
was  not  definitely  successful  until  1885,  when  Bumm  ^  obtained  growth 
upon  tubes  of  coagulated  human  blood  serum.  Bumm  was  not  only 
able  to  keep  the  organisms  alive  by  transplantation  in  pure  culture,  but 
produced  the  disease  by  inoculation  of  his  cultures  upon  the  healthy 
urethra. 

Morphology  and  Staining. — The  gonococcus  is  usually  seen  in  the 
diplococcus  form,  the  pairs  being  characteristically  flattened  along  the 
surfaces  facing  each  other.  This  gives  the  cocci  a  peculiar  coffee-bean 
or  biscuit  shape.  The  size  of  the  diploforms  is  about  1.6  micra  in  the 
long  diameter,  about  0.8  micron  in  width.  Stained  directly  in  gonorrheal 
pus  from  acute  cases,  the  microorganisms  are  found  both  intra-  and 
extracellularly,  a  large  number  of  them  crowded  characteristically 
within  the  leucocytes.  They  are  never  found  within  the  nucleus.  The 
phagocytosis. which  produces  this  picture  has  been  shown  by  Scholtz  ^ 
and  others  to  take  place  in  the  free  secretions,  not  in  the  depth  of  the 
tissues.  The  intracellular  position,  which  is  of  considerable  diagnostic 
importance,  is  lost  to  a  great  extent  in  secretions  from  chronic  cases. 
In  smears  made  from  pure  cultures  the  arrangement  in  groups  of  two 
may  often  be  less  marked  than  in  pus,  clusters  of  eight  or  more  being 
common. 

1  Neisser,  Cent.  f.  d.  med.  Wiss.,  1879. 

2  Bumm,  "  Beitr.  z.  Kenntniss  des  Gonococcus,"  Wiesbaden,  1885. 

3  Scholtz,  Arch,  f,  Dermat,,  1899. 

330 


DIPLOCOCCUS  GONORRHCEiE^ 


381 


The  gonococcus  is  non-motile  and  does  nof  form  spores.  It  is  easily 
stained  with  aqueous  anilin  dyes.  Methylene-blue  alone,  or  eosin 
followed  by  methylene-blue,  give  good  results.  An  excellent  picture 
is  obtained  with  the  Pappenheim-Saathof  stain  consisting  of 

Methyl  green 0 .  15 

Pyronin 0.5 

96%  alcohol 5.0 

•      Glycerin 20.0 

2%  carbolic  acid  water  ad  .• 100 . 0 

Fix  stain  1-2  min. 


Gram's  method  of  staining,  however,  is  the  only  one  of  differential 
value,  gonococcus  being  Gram  negative.  The  Gram  stain  applied  to 
pus  from  the  male  urethra,  while  not  absolutely 
reliable,  is,  for  practical  purposes,  sufficiently  so 
to  make  a  diagnosis.  In  exudates  from  the 
vagina  or  from  the  eye  the  morphological  pic- 
ture is  not  so  reliable,  owing  to  the  frequent 
presence  in  these  regions  of  other  Gram-negative 
cocci.  The  great  scarcity  of  gonococci  in  very 
chronic  discharges  necessitates  thorough  cultural 
investigation;  negative  morphological  examina- 
tion in  such  cases  can  not  be  regarded  as  con- 
clusive.^ 

Cultivation. — The  gonococcus  is  delicate  and 
difficult  to  cultivate.  Bumm  ^  obtained  his  first 
growths  upon  human  blood  serum  which  had  been 
heated  to  partial  coagulation. 

The  medium  most  commonly  used  at  the 
present  day  was  introduced  by  Wertheim,^  and 
consists  of  a  mixture  of  two  or  three  parts  of 
meat  infusion-agar  with  one  part  of  uncoagulated 
human  ascitic  fluid,  hydrocele  fluid,  or  blood 
serum.  The  agar  is  melted  and  cooled  to  45° 
is  added.  The  mixture  may  then  be  slanted 
or  poured  into  a  Petri  plate. 


Fig.  80. — Gonorrheal 
Pus  FROM  Urethra, 
Showing  the  Cocci 
WITHIN  A  Leucocyte. 


before  the  serum 
in  the  test  tube 
One  per  cent  of  glucose  may  be  added. 
Cultures  in  fluid  media  may  be  obtained  by  similar  additions  of  serum 
to  meat-infusion-pepton-broth.     Whole  rabbit's  blood  added  to  agar, 


^  Heinian,  Medical  Record,  1896.         "  Bumm,  Deut.  med.  Woch.,  1885. 
3  Wertheim,  Arch,  f .  Gynakol.,  1892. 


\.' 

•• 

% 

9       % 

4 

•  -'Z 

, 

•       • 

-•* 

• 

•  • 

'•  .••.■. 

•*  .%. 

•# 

..  ^■■.; 

/.• 

M 

• 

j                          • 

•  •• 

•  • 

.    y 

%    «• 

"T. 

••:.. 

i 

382  PATHOGENIC  MICROORGANISMS 

or  the  swine-serum-nutrose  medium  of  Wassermarin  ^  may  occasionally 

be  used  with  success. 

Plates  may  be  made  by  smearing  for  enrichment  a  drop  of  blood 

from  the  finger  over  the  surface  of  agar  in  the  manner  of  Pfeiffer's 

method  for  influenza-bacillus  cultivation.     Inoculations  from  gonorrheal 

material  are  best  made  by  surface 
smearing  upon  plates,  since  the  gono- 
coccus  grows  best  in  the  presence  of 
free  oxygen.  Growth  becomes  more 
luxuriant  after  prolonged  cultivation 
upon  artificial  media.  The  most 
favorable  reaction  of  media  is  neu- 
trality or  slight  acidity. 

When  the  gonococcus  has  been 
successfully  cultivated  from  pus  upon 
media  without  serum   additions,  the 

success  has  probably  been  due  to  the 
Fig.  81. — Gonococcus.     Smear  ,    ,  .    ,  •       -i 

f  ,,  substances  carried   over   in   the   pus. 

from  pure  culture.  i  .        .  . 

The  ease  of  cultivation  differs  con- 
siderably with  different  strains  of  gonococci.  Some  grow  very  heavily 
after  first  isolation,  but  the  majority  show  a  very  delicate  growth 
even  on  rich  ascitic  glucose  agar.  After  several  generations  of  growth 
on  artificial  media,  however,  the  organism  develops  with  increasing  ease 
and  on  simpler  media.  It  may  eventually  be  cultivated  on  plain  agar, 
especially  when  this  is  made  of  veal  infusion.  Recently  a  medium  upon 
which  gonococci  after  first  cultivation  can  be  grown  with  ease  has  been 
recommended  by  Edward  B.  Vedder.^  The  medium  consists  of  a 
1 . 5  to  1 .  75  per  cent  agar  made  with  beef  infusion  neutral  to  phenol- 
phthalein,  and  after  clearing,  1  per  cent  of  corn  starch  added.  The  corn 
starch  is  best  added  after  grinding  with  a  little  agar  to  avoid  clumps, 
this  then  being  poured  into  the  bulk  of  the  agar  and  thoroughly  mixed. 
The  medium  should  be  sterilized  at  not  over  15  lbs.  pressure  to  avoid 
changes  in  the  starch.  Recently  we  have  isolated  several  strains  of 
gonococci  which  grew  very  heavily  on  simple  media  without  ascitic 
fluid  in  the  second  culture  generation. 

^  Wassermann,  Berl.  klin.  Woch.,  1897. 

(Fifteen  c.c.  swine-serum,  35  c.c.  of  water,  3  c.c.  glycerin,  with  two  per  cent 
nutrose.  The  nutrose  is  dissolved  by  boiling  and  the  solution  sterilized.  This  is  then 
added  to  agar,  in  equal  parts,  and  used  in  plates.) 

2  Vedder,  Jour.  Infec.  Dis.,  May  15,  1915,  xvi,  385. 


DIPLOCOCCUS  GONORRHCE^ 


383 


The  gonococcus  will  develop  sparsely  under  anaerobic  conditions, 
but  has  marked  preference  for  aerobiosis.  The  optimum  temperature 
is  37.5°  C.     Growth  ceases  above  38.5°  and  below  30°. 

Upon  suitable  media  colonies  appear  as  extremely  delicate,  grayish, 
opalescent  spots,  at  the  end  of  twenty-four  hours.  The  separate  colo- 
nies do  not  tend  to  confluence 
and  have  slightly  undulated 
margins.  Touched  with  a 
platinum  loop  their  consist- 
ency is  found  to  be  slimy.  In 
fluid  media,  growth  takes 
place  chiefly  at  the  surface. 

Until  recent  years  the 
gonococci  were  regarded  as  a 
compact  group,  within  which 
individual  members,  isolated 
from  different  cases,  were 
identical  in  all  respects.  How- 
ever, work  done  by  Torrey,* 
and  by  Teague  and  Torrey,^ 
has  shown  that  immunologic- 
ally gonococci  can  be  divided 
into  a  number  of  different 
groups.    The  situation  here  is 

closely  analogous  to  that  which  has  developed  in  the  case  of  the  pneumo- 
coccus  group.  By  cross  agglutinations  and  agglutinin  absorption  expe- 
riments, Torrey  has  been  able  to  show  that  gonococci  fall  into  about  10 
groups,  A,  B,  C,  G,  K,  L,  N,  O,  Q,  S,  which  are  serologically  separable  one 
from  the  other.  The  practical  importance  of  this  is  that  in  all  diagnostic 
serum  reactions,  such  as  the  complement-fixation  test,  it  is  necessary 
to  make  a  polyvalent  antigen  in  which  these  various  groups  shall  be 
represented.  Otherwise,  of  course,  many  cases  would  react  negatively 
if  infected  with  a  particular  group  which  is  not  included  in  the  antigen. 

Resistance. — Recent  cultures  of  gonococcus,  if  not  transplanted, 
usually  die  out  within  five  or  six  days  at  incubator  temperature.  At 
room  temperature  they  die  more  rapidly. 

The  resistance  of  the  gonococcus  to  light  and  heat  is  slight.  A  tem- 
perature of  41°  to  42°  kills  it  after  a  brief  exposure.    Complete  drying 

1  Torrey,  Jour,  of  Med.  Research,  1907,  xvi,  329. 

2  Teague  and  Torrey,  Jour,  of  Med.  Research,  Dec,  1907,  xvii,  223. 


Fig.  82. — Gonococcus  Colony.     Low  power 
of  magnification.   (After  Mallory  and  Wright.) 


384  PATHOGENIC  MICROORGANISMS 

destroys  it  in  a  short  time.  Incompletely  dried,  however,  and  pro- 
tected from  light  (gonorrheal  pus)  it  may  live,  on  sheets  and  clothing, 
for  as  long  as  eighteen  to  twenty-four  hours.^ 

It  is  easily  killed  by  most  disinfectant  solutions  ^  in  high  dilution 
and  seems  to  be  almost  specifically  sensitive  to  the  \arious  silver  salts, 
a  fact  of  therapeutic  importance. 

Pathogenicity. — Gonorrheal  infection  occurs  spontaneously  only  in 
man.  True  gonorrheal  urethritis  has  never  been  experimentally  pro- 
duced in  animals.  In  human  beings,  apart  from  the  infection  in  the 
male  and  female  genital  tracts,  and  in  the  conjunctivae,  the  gonococcus 
may  produce  cystitis,  proctitis,  and  stomatitis.  It  may  enter  the  cir- 
culation, giving  rise  to  septicemia,  ^  to  endocarditis  and  arthritis.  Iso- 
lated cases  of  gonorrheal  periostitis  and  osteomyelitis  have  been  re- 
ported.^ 

The  acute  infections  of  the  genito-urinary  passages  are  often  fol- 
lowed by  prolonged  chronic  infection,  which,  though  quiescent,  may  for 
many  years  be  a  source  of  social  danger.  In  children,  especially  females, 
the  infection  is  not  rare,  and  may  assume  epidemic  characters,  traveling 
from  bed  to  bed  in  institutions.  Such  hospital  epidemics  can  be  stopped 
only  by  the  most  rigid  isolation.  It  is  advisable,  in  view  of  this  danger, 
^to  examine  all  female  children  applying  for  admission  to  a  hospital,  by 
'vaginal  smear  and,  if  possible,  to  keep  them  in  a  receiving  ward  for 
twenty-four  hours  in  order  that  the  examination  may  be  repeated  before 
admission  to  the  general  wards.  In  the  best-equipped  institutions,  fur- 
thermore, separate  thermometers,  bed  linen,  and  diapers  are  set  aside 
for  each  child  in  order  to  preclude  any  possibility  of  accidental  transmis- 
sion from  cases  which  may  have  escaped  detection  by  smear  examination. 

While  inoculation  of  animals  has  never  resulted  in  active  prolifera- 
tion of  the  gonococcus  upon  the  new  host,  local  necrosis,  suppuration, 
and  temporary  systemic  reactions  have  been  produced  by  subcutaneous 
and  intraperitoneal  inoculation.  A  toxin  has  been  isolated  by  Niko- 
laysen  ^  by  extraction  from  the  bacterial  bodies  with  distilled  water  or 
sodium  hydrate  solutions.  It  was  found  to  be  resistant  to  a  tempera- 
ture of  120°  and  to  remain  potent  after  complete  drying.  The  same 
author  found  that  the  isolated  toxin  and  dead  cultures  were  fully  as 
toxic  for  animals  as  living  cultures,  0.01  gram  killing  a  white  mouse. 

^  Heiman,  Medical  Record,  1896. 

2  Schaeffer  und  Steinschneider,  Kong.  Deut.  Dermat.  Gesells.,  Breslau,  1894. 

'  Review  of  cases  of  Gon.  Septicemia,  Faure-Beaulieu,  Thesis,  Paris,  1906. 

*  ^Jllmann,  Wien,  med.  Presse,  1900.  ^  Nikolaysen,  Cent.  f.  Bakt.,  1897. 


1I4ICR0C0CCUS  CATARRHALIS  385 

Specific  injury  to  the  nervous  system  by  injections  of  gonococcus 
toxin  has  been  reported  by  Moltschanoff.^ 

The  secretion  of  a  true  soluble  toxin  by  the  gonococcus,  asserted  by 
Christmas,^  is  denied  by  Wassermann,^  Nikolaysen,^  and  others.  Christ- 
mas,^ and,  more  recently,  Torrey,^  hare  reported  successful  active  im- 
munization of  animals  by  repeated  injections  of  whole  bacteria.  Torrey 
and  others  apparently  have  successfully  treated  human  cases  by  injec- 
tions of  the  serum  of  immunized  animals. 

Antibodies  to  Gonococcus. —  Patients  infected  with  gonococci 
seem  to  produce  antibodies  against  the  organisms.  Although  in  the  or- 
dinary gonorrheal  urethritis,  or  vaginitis,  it  is  relatively  simple  to  make 
the  diagnosis  by  finding  gonococci  in  the  discharges,  diagnosis  may  be 
difficult  in  cases  of  gonorrheal  rheumatism,  or  endocarditis,  when  iso- 
lation of  the  bacteria  fails  or  when  the  connection  between  the  local 
venereal  disease  and  the  general  condition  is  obscure.  Various  sero- 
logical diagnostic  methods  have  been  attempted,  and  of  recent  years 
the  complement-fixation  test  has  been  found  to  be  very  useful.  The 
method  has  been  especially  developed  by  Archibald  McNeil,  at  the 
New  York  Department  of  Health.  It  consists  in  making  a  polyvalent 
antigen,  using  the  10  Torrey  strains  which  are  kept  in  stock  transplants 
on  glucose  ascitic  agar.  It  has  been  found  that  the  best  medium  for 
antigen  production  is  an  agar  made  of  "bob  veal."  For  the  production 
of  antigen,  stock  cultures  are  transplanted  on  "bob  veal"  agar,  without 
salt,  glucose  or  ascitic  fluid,  the  reaction  carefully  adjusted  to  an  acidity 
of  0.1  per  cent  to  0.2  per  cent.  Twenty-four  hour  growths  on  this  me- 
dium are  scraped  off  and  emulsified  in  neutral  sterile  distilled  water. 
The  emulsion  is  autolyzed  one  hour  in  a  water  bath  at  56°  and  heated 
one  hour  at  80°  C.  It  is  then  filtered  through  a  sterile  Berkfeld  filter. 
The  filtrate  is  aseptically  bottled  and  sterilized  3  days  at  56°,  half  an 
hour  each  day.    It  is  then  made  isotonic  and  is  ready  for  titration. 

Vaccine  therapy  in  systemic  gonorrheal  infection  has  been  tried  and  is 
reasonably  successful.  The  vaccine,  if  possible,  should  be  made  with 
the  organism  isolated  from  the  patient,  for  reasons  described  above. 
Passive  immunization  with  the  serum  of  gonococcus-immune  animals 
has  also  been  attempted,  but  records  on  it  at  present  are  not  sufficiently 
complete  to  permit  definite  judgment.  ';:.' 

1  Moltschanoff,  Munch,  med.  Woch.,  1899,     ^Christmas,  Ann.  de  Tlnst. Pasteur,  1897. 
3  Wassermann,  Zeit,  f,  Hyg.,  xxvii,  1897.       *  Nikolaysen,  Fort.  d.  Med.,  xxi,  1897. 
^  Christmas,  loc,  cit. 
*  Torrey,  Jour.  Amer.  Med.  Assn.,  xlvi,  1906. 


386  PATHOGENIC  MICROORGANISMS 


MICROCOCCUS  CATARRHALIS 

Micrococcus  catarrhalis  is  a  diplococcus  described  first  by  R.  Pfeiffer*, 
who  found  it  in  the  sputum  of  patients  suffering  from  catarrhal  in- 
flammations of  the  upper  respiratory  tract.  It  was  subsequently  care- 
fully studied  by  Ghon  and  H.  Pfeiffer.^  According  to  these  authors 
the  pathogenic  significance  of  the  micrococcus  is  slight,  though  occasion- 
ally it  may  be  regarded  as  the  causative  factor  in  catarrhal  inflammations. 
Its  chief  claim  to  attention,  however,  lies  in  its  similarity  to  the  meningo- 
coccus and  the  gonococcus,  from  neither  of  which  it  can  be  morphologi- 
cally distinguished.  It  is  decolorized  by  Gram's  stain,  appears  often  in 
the  diplococcus  form,  and  has  a  tendency,  in  exudates,  to  be  located 
intracellularly.  Not  unlike  the  two  microorganisms  mentioned,  too,  it 
shows  but  slight  pathogenicity  for  animals. 

Differentiation  from  gonococcus  i§  extremely  simple  in  that  Micro- 
coccus catarrhalis  grows  easily  on  simple  culture  media  and  shows 
none  of  the  fastidious  cultural  requirements  of  the  gonococcus. 

From  meningococcus  the  differentiation  is  less  simple  and,  because 
of  the  presence  of  both  microorganisms  in  the  nose,  is  of  great  importance. 

Distinction  between  the  two  is  made  entirely  upon  cultural  charac- 
teristics and  agglutination  reactions.  Culturally,  Micrococcus  catar- 
rhalis grows  more  heavily  than  meningococcus  upon  the  ordinary 
culture  media.  The  colonies  of  Micrococcus  catarrhalis  are  coarsely 
granular  and  distinctly  white  in  contradistinction  to  the  finely  granu- 
lar, grayish  meningococcus  colonies.^  Micrococcus  catarrhalis  will 
develop  at  temperatures  below  20°  C,  while  meningococcus  will  not 
grow  at  temperatures  below  25°  C* 

Dunham,^  who  has  recently  made  a  comparative  study  of  meningo- 
coccus and  other  Gram-negative  diplococci  from  the  nose  and  throat, 
states  that  while  some  of  the  supposed  Micrococcus  catarrhalis  cul- 
tures are  easily  distinguished  from  meningococcus  simply  by  the  char- 
acteristics of  their  growths  upon  two-per-cent  glucose  agar,  others  offer 
great  difficulties  to  differentiation.  He  recommends  as  a  differential 
medium  a  mixture  of  sheep  serum  and  bouillon  containing  1%  of  glucose. 
Upon  this  medium  all  true  meningococci  produce  acid,  but  no  coagulation, 


»  Flugge,  "Die  Mikroorg.,"  3d  ed.,  1896. 

«  Ghxm  und  H.  Pfeiffer,  Zeit.  f.  klin.  Med.,  1902.    »  Ghon  und  Pfeiffer,  loc.  cit. 

*  Weichselbaum,  in  Kolle  und  Wassermann,  Bd.  iii,  p.  269. 

^  Dunham,  Jour.  Inf.  Dis..  190'^ 


GRAM-NEGATIVE  COCCI 


387 


with  24  hours.     Cultures  from  the  nose  and  throat,  however,  produce 
acid  and  coagulation,  or  else  produce  an  alkaline  reaction. 

OTHER  GRAM-NEGATIVE  COCCI 

Micrococcus  pharyngis  siccus. — First  described  by  von  Lingelsheim^ 
in  1906.  It  is  described  by  Elser  and  Huntoon  as  readily  differentiable 
from  meningococcus  and  other  Gram-negative  cocci  by  the  firm  adher- 
ence and  dryness  of  its  colonies.  It  is  similar  to  Micrococcus  catarrhalis 
from  which  it  may,  however,  be  differentiated  by  fermentation  tests. 

Diplococcus  mucosus. — This  organism  was  also  described  by  von 
Lingelsheim  together  with  the  preceding  one.  Its  colony  formation  is 
similar  to  that  of  meningococcus,  but  slightly  more  sticky  and  mucoid. 
Stained  by  the  capsule  methods,  it  is  seen  to  possess  a  distinct  capsule. 

Chromogenic  Gram-negative  Cocci. — These  microorganisms  all  pro- 
duce a  greenish-yellow  pigment  on  the  ordinary  culture  media.  When 
pigment  is  absent,  as  is  frequently  the  case  when  grown  upon  sugar-free 
media,  these  microorganisms  can  be  distinguished  from  meningococcus 
only  by  sugar  fermentation  and  senrni  reactions. 

An  exhaustive  study  of  Gram-negative  micrococci  has  recently  been 
made  by  Elser  and  Huntoon.^  These  authors,  in  studying  the  differ- 
ential value  of  sugar  fermentation  in  the  diagnosis  of  these  bacteria, 
have  constructed  the  following  table: 


Strains  Tested 

Meningococcus 

Pseudomeningococcus 

Gonococcus 

Micrococcus  catarrhalis 

Micrococcus  pharyngis  siccus 

Chromogenic  group  I 

Chromogenic  group  II 

Chromogenic  group  III 

Jaeger  meningococcus,  Krai. . 
Diplococcus  crassus,  Krai 


Strains 

Dex- 

Malt- 

Levu- 

Saccha- 

Lac- 

trose 

ose 

lose 

rose 

tose 

200 

+ 

+ 

0 

0 

0 

6 

+ 

+ 

0 

0 

0 

15 

+ 

0 

0 

0 

0 

64 

0 

0 

0 

0 

0 

2 

+ 

+ 

+ 

0 

28 

+ 

+ 

+ 

0 

11 

+ 

+ 

+ 

0. 

0 

9 

+ 

+ 

0 

0 

0 

1 

+ 

+ 

+ 

+ 

+ 

1 

+ 

+ 

+ 

+ 

+ 

Gal- 
actose 


1 V.  Ldngelsheim,  Klin.  Jahrb.,  15, 1906, 


*  EUer  and  Huntoon,  loc.  cit. 


CHAPTER  XXVI 
BACILLI  OF  THE  COLON-TYPHOID-DYSENTERY  GROUP 

The  bacilli  belonging  to  this  group  of  microorganisms/while  present- 
ing great  differences  in  their  pathogenic  characteristics,  possess  many 
points  of  morphological  and  biological  similarity  which  have  made  their 
differentiation  extremely  difficult.  Among  pathogenic  bacilli,  they  are 
probably  the  ones  most  commonly  encountered  and  because  of  the  fact 
that  some  of  them  are  specifically  pathogenic,  while  others  are  essen- 
tially saprophytic  and  are  pathogenic  only  under  exceptional  conditions, 
the  necessity  of  accurate  differentiation  is  a  daily  occurrence  in  bacteri- 
ological laboratories.  It  has  been  through  the  study  of  this  group  par- 
ticularly that  many  of  the  modern  differential  methods  of  bacteriology 
have  been  developed. 

The  group  includes  the  colon  bacillus  and  its  allies,  the  typhoid 
bacillus,  the  paratyphoid  organisms,  the  several  varieties  of  dysentery 
bacillus  and  numerous  closely  related  species,  and  Bacillus  fecalis  alka- 
ligenes.  Closely  related  to  the  group  though  not  properly  within  it, 
are  Bacillus  lactis  aerogenes,  B.  acidi  lactici  bacilli  of  the  Friedlander  or 
mucosus  capsulatus  group,  and  a  number  of  less  important  subdivisions 
of  this  last  group. 

All  bacilli  of  the  group  possess  morphological  characteristics  which^ 
although  exhibiting  slight  differences,  are  insufficient  to  permit  accurate 
morphological  diagnosis.  They  are  none  of  them  spore-bearing.  Stained 
by  Gram's  method  they  are  decolorized. 

Cultivated  upon  artiftcial  media,  they  grow  readily  both  at  room  and 
at  incubator  temperatures.  None  of  them  liquefies  gelatin.  Though 
showing,  often,  distinct  differences  in  the  speed  and  luxuriance  of  growth 
upon  ordinary  media,  these  differences  are,  nevertheless,  too  slight  to 
become  the  basis  of  differentiation. 

In  order  to  distinguish  between  the  individual  members  of  this 
group,  therefore,  we  are  forced  to  a  careful  biological  and  cultural 
study.  This  is  carried  out  by  the  observation  of  the  cultural  character- 
istics upon  special  media  and  by  the  study  of  serum  reactions  in  speci- 
fic immune  sera.    Our  mainstays  in  the  accurate  differentiation  of  these 

388 


BACILLUS  COLl  COMMUNIS  389 

bacilli  are  the  observation  of  their  fermentative  action  upon  carbohy- 
drate media,  and  their  agglutinating  reactions  in  immune  sera.  These 
'points  will  be  alluded  to  in  the  description  of  the  individual  microor- 
ganisms, and  will  be  again  summarized  in  the  differential  tables  given 
at  the  end  of  the  chapters  dealing  with  this  group. 

BACILLUS    GOLI    COMMUNIS    AND    MEMBERS    OF    THE    COLON 

BACILLUS   GROUP 

Under  the  name  of  "  colon  bacilli "  are  grouped  a  number  of  bac- 
terial varieties  differing  from  one  another  somewhat  in  minor  character- 
istics, but  corresponding  in  certain  cardinal  points  which  stamp  them 
as  close  relatives  and  amply  warrant  their  consideration  under  one 
heading.  While  usually  Hving  as  harmless  parasites  upon  the  animal 
and  human  body,  and  capable  of  leading  a  purely  saprophytic  existence, 
they  may,  nevertheless,  under  certain  circumstances  become  pathogenic 
and  thus,  both  culturally  and  in  their  pathological  significance,  form  a 
link  between  pure  saprophytes  like  Bacillus  lactis  aerogenes,  on  the 
one  hand,  and  the  more  strictly  pathogenic  Gram-negative  bacilli  of 
the  paratyphoid,  typhoid,  and  dysentery  groups,  on  the  other.  As  a 
type  of  the  group  we  may  consider  in  detail  its  most  prominent  and 
thoroughly  studied  member,  Bacillus  coli  communis. . 

BACILLUS   COLI   COMMUNIS 

This  microorganism  was  seen  and  described  by  Buchner  ^  in  1885. 
It  was  thoroughly  studied  in  the  years  immediately  following,  especially 
by  Escherich,^  in  connection  with  the  intestinal  contents  of  infants. 

Morphology. — Bacillus  coli  communis  is  a  short,  plump  rod  about 
1-3  micra  long,  and  varying  in  thickness  from  one-third  to  one-fifth 
of  its  length.  Under  varying  conditions  of  cultivation,  it  may  appear 
to  be  more  slender  than  this  or  shorter  and  even  coccoid  in  form.  In 
stained  preparations,  it  usually  appears  singly,  but  occasionally  may  be 
seen  in  short  chains.  It  stains  readily  with  the  usual  anilin  dyes  and 
decolorizes  by  Gram's  method.  Spores  are  not  formed.  It  is  motile,  and 
flagella  staining  reveals  eight  or  more  flagella  peripherally  arranged.  Its 
motility  is  subject  to  wide  variations.    Young  cultures,  in  the  first  gen- 

1  Buchner,  Arch.  f.  Hyg.,  3,  1885. 

^Escherich,  "Die  Darmbakt.  des  Sauglings,"  Stuttgart,  1880;  Cent.  f.  Bakt.,  1, 

1887. 


390 


PATHOGENIC  MICROORGANISMS 


eration,  after  Isolation  from  the  body,  may  be  extremely  motile,  while  old 
laboratory  strains  may  show  almost  no  motility.  Independent  of  these 
modifying  conditions,  however,  separate  races  may  show  individual 
characteristics  as  to  motility,  varying  in  range  between  a  motility 
hardly  distinguishable  from  Brownian  movement  and  one  which  is  so 
active  as  to  be  but  little  less  than  that  of  the  typhoid  bacillus.  Ordi- 
narily, the  colon  bacillus  possesses  a  motility  intermediate  between 
these  two  extremes. 

Cultivation. — The  bacillus  is  an  aerobe  capable  of  anaerobic  growth 
under  suitable  cultural  conditions.    It  grows  well  on  the  simplest  media 


i^iG.  83. — Bacillus  coli  communis. 


at  temperatures  ranging  from  20°  to  40°  C,  but  finds  its  optimum 
growth  at  about  37.5°  C.  Upon  broth  it  grows  rapidly,  giving  rise  to 
general  clouding;  later  to  a  pgllicle  and  a  light,  slightly  slimy  sediment. 
Withm  moderate  ranges,  it  is  not  delicately  susceptible  to  reaction, 
growing  equally  well  on  media  slightly  acid  and  on  those  of  a  moderate 
alkalinity. 

Upon  agar,  it  forms  grayish  colonies  which  become  visible  within 
twelve  to  eighteen  hours,  gradually  becoming  more  and  more  opaque 
as  they  grow  older.    The  deep  colonies  are  dense,  evenly  granular,  oval. 


BACILLU&  COLI  COMMUNIS  391 

or  round.  Surface  colonies  often  show  a  characteristic  grape-leaf 
structure,  or  may  be  round  and  flat,  and  show  a  definitely  raised,  glisten- 
ing surface.    Upon  agar  slants,  growth  occurs  in  a  uniform  layer. 

On  gelatin  the  colon  bacillus  grows  rapidly,  causing  no  liquefaction. 
Surface  colonies  are  apt  to  show  the  typical  grape-leaf  formation.  Deep 
colonies  are  round,  oblong,  and  glistening.  In  gelatin  stabs  growth  takes 
place  along  the  entire  line  of  inoculation,  spreading  in  a  thin  layer  over 
the  surface  of  the  medium. 

On  potato,  growth  is  abundant  and  easily  visible  within  eighteen 
to  twenty-four  hours,  as  a  grayish-white,  glistening  layer  which  later 
turns  to  a  yellowish-brown,  and  in  old  cultures  often  to  a  dirty  green- 
ish-brown color. 

In  pepton  solution  indol  is  formed.  In  milk  there  is  acidity  and  co- 
agulation. In  lactose-litmus-agar  acid  is  formed,  the  medium  becom- 
ing red,  and  gas-bubbles  appear  along  the  line  of  the  stab  inoculation. 

In  carbohydrate  broth,  gas  is  formed  in  dextrose,  lactose,  and  mannit, 
but  not  in  saccharose.  Levulose,  galactose,  and  maltose  are  also  fer- 
mented with  the  formation  of  acid  and  gas. 

Cultures  of  the  colon  bacillus  are  characterized  by  a  peculiar  fetid 
odor  which  is  not  unlike  that  of  diluted  feces.  The  acids  formed  by  the 
colon  bacillus  from  sugars  are  chiefly  lactic,  acetic,  and  formic  acids. 
The  gas  it  produces  consists  chiefly  of  CO 2  and  hydrogen.  The  bacillus 
grows  well  on  media  containing  urine  and  on  those  containing  bile. 
Upon  the  latter  fact  some  methods  for  the  isolation  of  the  colon 
bacillus  from  water  and  feces  have  been  based. 

Isolation  of  the  colon  bacillus  from  mixed  cultures  is  most  easily 
accomplished  by  plating  upon  lactose-litmus-agar,  the  Conradi-Drigal- 
ski  medium,  or  the  Endo  medium  after  preliminary  enrichment  of  the. 
specimen  to  be  tested  in  bile  or  malachite-green  broth.  (In  the  case 
of  feces  such  enrichment  is  superfluous.) 

Distribution. — The  colon  bacillus  is  a  constant  inhabitant  of  the 
intestinal  canal  of  human  beings  and  animals.  It  is  also  found  occasion- 
ally in  soil,  in  air,  in  water,  and  in  milk  and  is  practically  ubiquitous  in 
all  neighborhoods  which  are  thickly  inhabited.  When  found  in  nature 
its  presence  is  generally  taken  to  be  an  indication  of  contamination  from 
human  or  animal  sources.  Thus,  when  found  in  water  or  milk,  much 
hygienic  importance  is  attached  to  it.  Recently,  Papasotiriu  ^  and, 
independently  of  him,  Prescott,^  have  reported  finding  bacilli  apparently 

» Papasotiriu,  Arch,  f,  Hyg.,  xU.     ^  Presoott,  Cent.  f.  B^kt ,  Ref.,  xxxiii,  1903, 


302  PATHOGENIC   MICROORGANISMS 

identical  with  Bacillus  coli  upon  rye,  barley,  and  other  grains.  They 
believCj  upon  the  basis  of  this  discovery,  that  Bacillus  coli  is  widely 
distributed  in  nature  and  that  its  presence,  unless  it  appears  in  large 
numbers,  does  not  necessarily  indicate  recent  fecal  contamination. 
These  reports,  however,  have  not  found  confirmation  by  the  work  of 
others,  and  can  not,  therefore,  be  as  yet  accepted. 

In  man.  Bacillus  coli  appears  in  the  intestine  normally  soon  after 
birth,  at  about  the  time  of  taking  the  first  nourishment.^  From  this  time 
on,  throughout  life,  the  bacillus  is  a  constant  intestinal  inhabitant  ap- 
parently without  dependence  upon  the  diet.  Its  distribution  within  the 
intestine,  according  to  Gushing  and  Livingood,^  is  not  uniform,  it  being 
found  in  the  greatest  numbers  at  or  about  the  ileocecal  valve,  diminish- 
ing from  this  point  upward  to  the  duodenum  and  downward  as  far  as 
the  rectum.  Adami  ^  and  others  claim  that,  under  normal  conditions, 
the  bacillus  may  invade  the  portal  circulation,  possibly  by  the  inter- 
mediation of  leucoeytic  emigration  during  digestion.  After  death,  at 
autopsy,  Bacillus  coli  is  often  found  in  the  tissues  and  the  blood  with- 
out there  being  visible  lesions  of  the  intestinal  mucous  membrane.^  It 
is  probable,  also,  that  it  may  enter  and  Hve  in  the  circulation  a  few 
hours  before  death  during  the  agonal  stages. 

Extensive  investigations  have  been  carried  out  to  determine  wheth- 
er or  not  the  constant  presence  of  this  microorganism  in  the  intestinal 
tract  is  an  indication  of  its  possessing  a  definite  physiological  function  of 
advantage  to  its  host.  It  has  been  argued  that  it  may  aid  in  the  fermen- 
tation of  carbohydrates.  The  question  has  been  approached  experiment- 
ally by  a  number  of  investigators.  Nuttall  and  Thierfelder  ®  delivered 
guinea-pigs  from  the  mother  by  Cesarean  section  and  succeeded  in 
keeping  them  without  infection  of  the  intestinal  canal  for  thirteen  days. 
Although  no  microorganisms  of  any  kind  were  found  in  the  feces  of 
these  animals,  no  harm  seemed  to  accrue  to  them,  and  some  of  them 
even  gained  in  weight.  Schottelius,^  on  the  other  hand,  obtained  con- 
tradictory results  when  working  with  chicks.  Allowing  eggs  to  hatch  in 
an  especially  constructed  glass  compartment,  he  succeeded  in  keeping  the 

1  Schild,  Zeit.  f.  Hyg.,  xix,  1895;  Lembke,  Arch.  f.  Hyg.,  xxvi,  1896. 

2  Gushing  and  Livingood,  "  Contributions  to  Med.  Sci.  by  Pupils  of  Wm.  Welch," 
Johns  Hopk.  Press,  1900. 

3  Adami,  Jour,  of  Amer.  Med.  Assn.,  Dec,  1899. 

*  Birch-Hirschfeld,  Ziegler's  Beitr.,  24,  1898. 

»  NiUtall  und  Thierfelder,  Zeit.  f.  Physiol.  Chemie,  xxi  and  xxii 

•  Schot^lius,  Arch.  f.  Hyg.,  xxxiv^  1889. 


BACILLUS  COLI  COMMUNIS  393 

chicks  and  their  entire  environment  sterile  for  seventeen  days.  During 
this  time  they  lost  weight,  did  not  thrive,  and  some  of  them  were  mori- 
bund at  the  end  of  the  second  week,  in  marked  contrast  to  the  healthy, 
well-noumished  controls,  fed  in  the  same  way,  but  under  ordinary  en- 
vironmental conditions.  Although  insufficient  work  has  been  done  upon 
this  important  question,  and  no  definite  statement  can  be  made,  it  is 
more  than  likely  that  the  function  of  the  Bacillus  coli  in  the  intestine 
is  not  inconsiderable  if  only  because  of  its  possible  antagonism  to  cer- 
tain putrefactive  bacteria,  a  fact  which  has  been  demonstrated  in  inter- 
esting studies  by  Bienstock  ^  and  others.^ 

Pathogenicity. — The  pathogenicity  of  the  colon  bacillus  for  animals 
is  slight  and  varies  greatly  with  different  strains.  Intraperitoneal  in- 
jections of  1  c.c.  or  more  of  a  broth  culture  will  often  cause  death  in 
guinea-pigs.  Large  doses  intravenously  administered  to  rabbits  may 
frequently  cause  a  rapid  sinking  of  the  temperature  and  death  with 
symptoms  of  violent  intoxication  within  twenty-four  to  forty-eight 
hours.  Subcutaneous  inoculation  of  moderate  doses  usually  results  in 
nothing  more  than  a  localized  abscess  from  which  the  animals  recover. 
It  is  likely  that,  even  in  fatal  cases,  death  results  chiefly  from  the  action 
of  poisons  liberated  from  the  disintegrating  bacteria  and  not  from  the 
multiplication  of  the  bacilli  themselves,  for  often  no  living  organism 
can  be  found  unless  large  doses  are  given. 

In  man,  a  large  variety  of  lesions  produced  by  Bacillus  coli  have 
been  described.  It  is  a  surprising  fact  that  disease  should  be  caused 
at  all,  in  man,  by  a  microorganism  which  is  so  constantly  present  in 
large  numbers  in  the  intestine  and  against  which,  therefore,  it  is  to  be 
expected  that  a  certain  amount  of  immunity  should  be  developed.  A 
number  of  explanations  for  this  state  of  affairs  have  been  advanced, 
none  of  them  entirely  satisfactory.  It  is  probable  that  none  of  the  poi- 
sonous products  of  the  colon  bacillus  is  absorbed  unchanged  by  the 
healthy  unbroken  mucosa  and  that,  therefore,  the  microorganisms  are, 
strictly  speaking,  at  all  times,  outside  of  the  body  proper.  Under  these 
circumstances,  no  process  of  immunization  would  be  anticipated.  It 
is  also  possible  that,  whenever  an  infection  with  Bacillus  coli  does  occur, 
the  infecting  organism  is  one  which  has  been  recently  acquired  from 
another  host,  having  no  specific  adaptation  to  the  infected  body.  Viru- 
lence may  possibly  be  enhanced  by  inflammatory  processes  caused  by 
other  organisms.    Considering  the  subject  from  another  point  of  view, 

»  Bienstock,  Arch.  f.  Hyg.,  xxxix,  190L  ^ 

a  Tissier  and  Martelly,  Ann.  de  I'inst.  Pasteur,  1902. 


394  PATHOGENIC  MICROORGANISMS 

colon-bacillus  infection  may  possibly  take  place  simply  because  of  unu- 
sual temporary  reduction  of  the  resistance  of  the  host.  Whether  or  not 
altered  cultural  conditions  in  the  intestine  may  lead  to  marked  enhance- 
ment in  the  virulence  of  the  colon  bacilli  can  not  at  present  be  decided. 
The  opinion  has  been  frequently  advanced,  however,  without  adequate 
experimental  support. 

Septicemia,  due  to  the  colon  bacillus,  has  been  described  by  a  large 
number  of  observers.  It  is  doubtful,  however,  whether  many  of  these 
cases  represent  an  actual  primary  invasion  of  the  circulation  by  the 
bacilli,  or  whether  their  entrance  was  not  simply  a  secondary  phenomenon 
occurring  during  the  agonal  stages  of  another  condition.  A  few  unques- 
tionable cases,  however,  have  been  reported,  and  there  can  be  no  doubt 
about  the  occurrence  of  the  condition,  although  it  is  probably  less 
frequent  than  formerly  supposed.  The  writers  have  observed  it  on 
two  occasions  in  cases  during  the  lethal  stages  of  severe  systemic 
disease  due  to  other  causes.  An  extremely  interesting  group  of  such 
cases  are  those  occurring  in  new-born  infants,  in  which  generalized 
colon-bacillus  infection  may  lead  to  a  fatal  condition  known  as 
WinckeFs  disease  or  hemorrhagic  septicemia.^  Prominent  among 
disease  processes  attributed  to  these  microorganisms  are  various  diar- 
rheal conditions,  such  as  cholera  nostras  and  cholera  infantum.  The 
relation  of  these  maladies  to  the .  colon  bacillus  has  been  studied  es- 
pecially by  Escherich,^  but  satisfactory  evidence  that  these  bacilli  may 
specifically  cause  such  conditions  has  not  been  brought.  While  it  is  not 
unlikely  that  under  conditions  of  an  excessive  carbohydrate  diet,  colon 
bacilli  may  aggravate  morbid  processes  by  a  voluminous  formation  of 
gas,  they  do  not,  of  themselves,  take  part  in  actual  putrefactive  proc- 
esses. It  is  likely,  therefore,  that  in  most  of  the  intestinal  diseases 
formerly  attributed  purely  to  bacilli  of  the  colon  group,  these  micro- 
organisms actually  play  but  a  secondary  part.^ 

It  is  equally  difficult  to  decide  whether  or  not  these  bacilli  may  be 
regarded  as  the  primary  cause  of  peritonitis  following  perforation  of 
the  gut.  Although  regularly  found  in  such  conditions,  they  are  hardly 
ever  found  in  pure  culture,  being  accompanied  usually  by  staphylococci, 
streptococci,  or  other  microorganisms,  whose  relationship  to  disease  is 
far  more  definitely  established.  Isolated  cases  have  been  reported, 
however,  one  of  them  by  Welch,   in  which  Bacillus  coli  was  present  in 

1  Kamen,  Ziegler's  Beitr.,  14,  1896. 

2  Escherich,  loc.  cit. 

8  Herter,  "  Bact.  Infec  of  Digest.  Tract,"  N.  Y.,  1907. 


BACILLUS  COLI  COMMUNIS  395 

the  peritoneum  in  pure  culture  without  there  having  been  any  intestinal 
perforation.*  Granting  that  the  bacillus  is  able  to  proliferate  within 
the  peritoneum,  there  is  no  reason  for  doubting  its  ability  of  giving  rise 
to  a  mild  suppurative  process. 

Infiammator}^  conditions  in  the  liver  and  gall-bladder  have  fre- 
quently been  attributed  to  the  colon  bacillus.  It  has  been  isolated  from 
liver  abscesses,  from  the  bile,  and  from  the  center  of  gall-stones.  Welch 
has  reported  a  case  of  acute  hemorrhagic  pancreatitis  in  which  the 
bacillus  was  isolated  from  the  gall-bladder  and  from  the  pancreas. 

In  the  bladder.  Bacillus  coli  frequently  gives  rise  to  cystitis  and  oc- 
casionally to  ascending  pyonephrosis.  No  other  microorganism,  in  fact, 
is  found  so  frequently  in  the  urine  as  this  one.  It  may  be  present,  often, 
in  individuals  in  whom  all  morbid  processes  are  absent.  The  condition 
is  frequently  observed  during  the  convalescence  from  typhoid  fever. 
It  may  disappear  spontaneously,  or  cystitis,  usually  of  a  mild,  chronic 
variety,  may  supervene. 

Localized  suppurations  due  to  this  bacillus  may  take  place  in  all 
parts  of  the  body.  They  are  most  frequently  localized  about  the  anus 
and  the  genitals.  They  are  usually  mild  and  easily  amenable  to  the 
simplest  surgical  treatment. 

Poisonous  Products  of  the  Colon  Bacillus. — The  colon  bacillus  belongs 
essentially  to  that  group  of  bacteria  whose  toxic  action  is  supposed  to 
be  due  to  the  poisonous  substances  contained  within  the  bacillary  body. 
Culture  filtrates  of  the  colon  bacillus  show  very  little  toxicity  when  in- 
jected into  animals;  whereas  the  injection  of  dead  bacilli  produces 
symptoms  almost  equal  in  severity  to  those  induced  by  injection  of  the 
live  microorganisms.  Corroborative  of  the  assumption  of  this  endotoxic 
nature  of  the  colon-bacillus  poison  is  the  fact  that,  so  far,  no  antitoxic 
bodies  have  been  demonstrated  in  serum  as  resulting  from  immuniza- 
tion. 

Immunization  with  the  Colon  Bacillus. — The  injection  into  animals  of 
gradually  increasing  doses  of  living  or  dead  colon  bacilli  gives  rise  to 
specific  bacteriolytic,  agglutinating,  and  precipitating  substances. 

The  bacteriolytic  substances  may  be  easily  demonstrated  by  the 
technique  of  the  Pfeiffer  reaction.  In  vitro  bacteriolysis  is  less  marked 
than  in  the  case  of  some  other  microorganisms  such  as  the  cholera  spiril- 
lum or  the  typhoid  bacillus.  Owing  probably  to  the  habitual  presence 
of  colon  bacilli  in  the  intestinal  tracts  of  animals  and  man,  considerable 

1  Welch,  Med.  News,  59,  1891. 


396 


PATHOGENIC  MICROORGANISMS 


bacteriolysis  may  occasionally  be  demonstrated  in  the  serum  of  normal 
individuals. 

Agglutinins  for  the  colon  bacillus  have  often  been  produced  in  the 
sera  of  immunized  animals  in  concentration  sufficient  to  be  active  in  dilu- 
tions of  1  :  5,000  and  over.  The  agglutinins  are  produced  equally  well 
by  the  injection  of  live  cultures  and  of  those  killed  by  heat,  if  the  tem- 
perature used  for  sterilization  does  not  exceed  100°  C.    It  is  *  a  notice- 


12  3 

Fig.  84. — Bacillus  coli  communis.  Grown  in:  1.  Dextrose,  2.  Lactose,  3. 
Saccharose  broth.  The  bacillus  forms  acid  and  gas  from  dextrose  and  lactose, 
not  from  saccharose.  Note  the  absence  of  growth  in  the  closed  arm  of  the  sac- 
charose tube,  in  which  no  acid  or  gas  is  formed. 

able  fact  that  the  injection  of  any  specific  race  of  colon  bacilli 
produces,  in  the  immunized  animal,  high  agglutination  values  only  for 
the  individual  culture  used  for  immunization,  while  other  strains  of 
colon  bacilli,  although  agglutinated  by  the  serum  in  higher  dilution 
than  are  paratyphoid  or  typhoid  bacilli,  require  much  higher  concen- 
tration than  does  the  original  strain.  The  subject  has  been  extensively 
studied  by  a  number  of  observers  and  illustrates  the  extreme  individual 


»  Wolff,  Cent.  f.  Bakt.,  xxv,  1899. 


BACILLUS  COLI  COMMUNIS 


397 


specificity  of  the  agglutination  reaction.  Thus  a  serum  which  will 
agglutinate  its  homologous  strains  in  dilutions  of  one  1 : 1,000  will  often 
fail  to  agglutinate  other  races  of  Bacillus  coli  in  dilutions  of  1:500 
or  1 :  600. 

The  normal  serum  of  adult  animals  and  man  will  often  agglutinate 
this  bacillus  in  dilutions  as  high  as  1 :  10  or  1 :  20 — a  phenomenon  pos- 
sibly referable  to  its  habitual  presence  within  the  body.  Corrobo- 
rating this  assumption  is  the  observation  of  Kraus  and  Low/ 
that  the  serum  of  new-born  animals  possesses  no  such  agglutinating 
powers.    The  fact  that  agglutinins  for  the  colon  bacillus  are  increased 


1  2 

Fig.  85. — Bacillus  coli  communior.     Grown  in: 

Saccharose  broth. 


1.  Dextrose,    2.  Lactose,    3, 


in  the  serum  of  patients  convalescing  from  typhoid  fever  or  dysentery 
is  probably  to  be  explained,  partly  by  the  increase  of  the  group  agglu- 
tinins produced  by  the  specific  infecting  ageiit,  and  partly  by  the  in- 
vasion of  colon  bacilli,  or  the  absorption  of  its  products  induced  by  the 
diseased  state  of  the  intestinal  mucous  membrane. 

Varieties  of  the  Colon  Bacillus. — During  the  earlier  days  of  bacterio- 
logical investigations,  a  large  number  of  distinct  varieties  of  colon  bacilli 
Were  described,  many  of  which  may  now  be  dismissed  as  based  simply 

1  Kraus  und  Low,  Wien.  klin.  Wocb.,  X899, 


398  PATHOGENIC   MICROORGANISMS 

upon  a  temporary  depression  of  one  or  another  cultural  characteristic  of 
Bacillus  coli  communis,  while  others  can  be  definitely  included  within 
other  closely  related,  but  distinct  groups. 

That  secondary  features,  such  as  dimensions,  motility,  and  luxuri- 
ance of  growth  upon  various  media,  may  be  markedly  altered  by  arti- 
ficial cultivation  is  a  common  observation.  It  has  not,  however,  been 
satisfactorily  shown  that  cardinal  characteristics,  such  as  the  forma- 
tion of  indol  from  pepton,  or  the  power  to  produce  gas  from  dextrose 
and  lactose,  can  be  permanently  suppressed  without  actual  injury  or 
inhibition  of  the  normal  vitality  of  the  microorganism.  Such  alter- 
ation is,  in  fact,  contrary  to  experience,  which  demonstrates  that 
whenever  such  changes  do  occur,  they  are  purely  temporary  and  a  few 
generations  of  cultivation  under  favorable  environmental  conditions 
will  regularly  restore  the  organism  to  its  normal  activity. 

Bacillus  coli  communior. — Distinct  and  constant  varieties  of  Bacil- 
lus coli,  however,  do  occur.  The  most  common  of  these  is  one  whicb 
Dunham  has  named  Bacillus  coli  communior,  because  of  the  -fact  that 
he  believes  it  to  be  more  abundant  4n  the  human  and  animal  intestine 
than  is  coli  communis  itself.  This  bacillus  possesses  all  the  cardinal 
characteristics  of  the  colon  group.  It  is  a  Gram-negative  bacillus, 
moderately  motile,  non-sporulating,  and  morphologically  indistinguish- 
able from  the  communis  variety.  It  does  not  liquefy  gelatin,  it 
produces  indol  from  pepton,  coagulates  and  acidifies  milk,  and  grows 
characteristically  upon. agar  and  potato.  It  differs  from  Bacillus  coli 
communis  in  that  it  produces  acid  and  gas  from  saccharose  as  well  as 
from  dextrose  and  lactose,  whereas  the  former  does  not  form  acid  or 
gas  from  saccharose. 


CHAPTER  XXVII 

BACILLI  OF  THE  COLON-TYPHOID-DYSENTERY  GROUP 

(Continued) 

THE   BACILLUS   OF   TYPHOID   FEVER 

{BacUlus  typhosus,  Bacillus  typhi  abdominalis) 

Typhoid  fever,  because  of  its  wide  distribution  and  almost  con- 
stant presence  in  most  communities,  has  from  the  eariiest  days  been  the 
subject  of  much  etiological  inquiry.  A  definite  conception  as  to  its 
infectiousness  and  transmission  from  case  to  case  was  formed  as  early 
as  1856  by  Budd.^ 

But  it  was  not  until  1880  that  Eberth  ^  discovered  in  the  spleen  and 
mesenteric  glands  of  typhoid- fever  patients  who  had  come  to  autopsy, 
a  bacillus  which  we  now  know  to  be  the  cause  of  the  disease.  Final 
proof  of  such  an  etiological  connection  was  then  brought  by  Gaffky,^ 
who  not  only  saw  the  bacteria  referred  to  by  Eberth,  but  succeeded  in 
obtaining  them  in  pure  culture  and  studying  their  growth  characteristics. 

Morphology  and  Staining. — The  typhoid  bacillus  is  a  short  rod  from 
1-3.5  /i  in  length  with  a  varying  width  of  from  .5  to  .8  At.  In  appear- 
ance it  has  nothing  absolutely  distinctive  which  could  serve  to  differen- 
tiate it  from  other  bacilli  of  the  typhoid-colon  group,  except  that  it  has 
a  general  tendency  to  greater  slenderness.  Its  ends  are  rounded  without 
ever  being  club-shaped.  Contrary  to  the  descriptions  of  the  earliest 
observers,  typhoid  bacilli  do  not  form  spores.  They  are  actively  motile 
and  have  twelve  or  more  flagella  peripherally  arranged. 

The  bacilli  stain  readily  with  the  usual  anilin  dyes.  Stained  by 
Gram's  method,  they  are  decolorized. 

Cultivation. — Bacillus  typhosus  is  easily  cultivated  upon  the  usual 
laboratory  media.  It  is  not  delicately  susceptible  to  reaction,  but  will 
grow  well  upon  media  moderately  alkaline  or  acid.  It  is  an  aerobic  and 
facultative  anaerobic  organism,  when  the  proper  nutriment  is  present. 
Upon  agar  plates  growth  appears  within  eighteen  to  twenty-four  hours 

»  Budd,  "  Intestinal  Fever/'  Lancet,  1856.  _ 

2  Eberth,  Virch,  Archiv,  81,  1880,  and  83,  1881. 
»  Gaffky,  Mitt.  a.  d.  kais,  Gesimdheitsamt,  2,  1884. 
399      . 


400 


PATHOGENIC  MICROORGANISMS 


as  small  grayish  colonies  at  first  transparent,  later  opaque.  Upon  agar 
slants  growth  takes  place  in  a  uniform  layer.  There  is  nothing  charac- 
teristic about  this  growth  to  aid  in  differentiation. 

In  broth,  the  typhoid  bacillus  grows  rapidly,  giving  rise  to  an  even 
clouding,  rarely  to  a  pellicle. 

Upon  gelatin,  the  typhoid  bacillus  grows  readily  and  does  not  lique- 
fy the  medium.  In  stabs,  growth  takes  place  along  the  entire  extent 
of  the  stab  and  over  the  surface  of  the  gelatin  in  a  thin  layer.  In  geW 
tin  plates  the  growth  may  show  some  distinction  from  that  of  other  mem- 
bers of  this  group,  and  this  medium  was  formerly  much  used  for  isolation 


Fig.  86. — Bacillus  typhosus,  from  twenty-four-hour  culture  on  agar. 


of  the  bacillus  from  mixed  cultures.  Growth  appears  within  twenty- 
four  hours  as  small,  transparent,  oval,  round,  or  occasionally  leaf-shaped 
colonies  which  are  smaller,  more  delicate,  and  more  transparent  than 
contemporary  colonies  of  the  colon  bacillus.  They  do  not,  however, 
show  any  reliable  differential  features  from  bacilli  of  the  dysentery 
group.  As  the  colonies  grow  older  they  grow  heavier,  more  opaque,  and 
lose  much  of  their  early  differential  value.  ^:    \  v 

On  potato  the  growth  of  typhoid  bacilli  is  distinctive,  and  this  medium 
was  recommended  by  Gaffky  ^  in  his  early  researches  for  purposes  of 

^Gaffky,\oc.  cit. 


BACILLUS  OF  TYPHOID  FEVER 


401 


identification.  On  it  typhoid  bacilli,  after  twenty-four  to  forty-eight 
hours,  produce  a  hardly  visible  growth,  evident  to  the  naked  eye 
only  by  a  slight  moist  glistening,  an  appearance  which  is  in  marked 
contrast  to  the  grayish-yellow  or  even  brown  and  abundant  growth 
of  the  colon  bacilli.  If  the  potato  medium  is  rendered  neutral  or 
alkaline,  this  distinction  disappears,  the  typhoid  bacillus  growing 
more  abundantly. 

In  milk,  typhoid  bacilli  do  not  produce  coagulation.  In  litmus-milk, 
during  the  first  twenty-four  hours,  the  color  is  changed  to  a  reddish  or 
violet  tinge  by  the  formation  of  acid  from  the  small  quantities  of  mono- 


FiG.  87. — Bacillus  typhosus,  showing  flagella.    (After  Frankel  and  Pfeiffer.) 


saccharid  present.  Later  the  color  becomes  deep  blue  from  the  forma- 
tion of  alkali. 

In  Dunham's  pepton  solution  no  indol  is  produced.  According  to 
Peckham,  however,  continuous  cultivation  in  rich  pepton  medi^  may 
lead  to  eventual  indol  formation  by  typhoid  bacilli.  This  fact  appears 
to  have  no  bearing  on  the  value  of  the  indol  test,  as  indol  is  never  formed 
under  the  usual  cultural  conditions. 

In  dextrose,  mannite,  lactose,  and  saccharose  broth,  the  typhoid  bacil- 
lus produces  no  gas.  A  comparative  summary  of  the  action  of  other 
bacilli  of  this  group  in  these  sugar  media  will  be  given  in  the  final  dif- 
ferential table  on  page  443. 


402 


PATHOGENIC  MICROORGANISMS 


f^ 


Tested  for  its  power  to  form  acid  from  sugars  commonly  used  in 
differential  tests,  typhoid  bacilli  form  acid,  but  no  gas,  on  the  mono- 
saccharides, on  mannit,  maltose  and  dextrin,  but  not  on  lactose  and 
saccharose.     (See  Table,  p.  44.) 

In  the  Hiss  tube  medium  (see  section  on  Media,  page  133),  the 
typhoid  bacillus  within  eighteen  to  twenty-four  hours  produces  an  even 
clouding  by  virtue  of  its  motility,  but  does  not  form  gas.  In  contradis- 
tinction to  this,  dysentery  bacilli  grow  only  along  the  line  of  inocula- 


FiG.  88. — Surface  Colony  of  Bacillus  typhosus  on  Gelatin.     (After  Heim.) 


tion,  while  bacilli  of  the  colon  group  move  in  irregular  sky-rocket-like 
figures  away  from  the  stab,  at  the  same  time  breaking  up  the  medium 
by  the  formation  of  gas-bubbles.  Some  actively  motile  colon  bacilli 
cloud  the  medium,  but  the  ruptures  caused  by  the  gas  are  always 
evident. 

The  differentiation  of  the  typhoid  bacillus  in  pure  culture  from  similar 
microorganisms  by  means  of  its  growth  upon  media  has  been  the  sub- 
ject of  many  investigations.  It  is  neither  practicable  nor  desirable  to 
enumerate  all  the  various  media  which  have  been  devised  and  reported. 


BACILLUS  OF  TYPHOID  FEVER  403 

The  aim  has  been  chiefly  the  differentiation  of  typhoid  bacilli  from  the 
bacilli  of  the  colon  group,  and  most  of  the  media  have  been  devised  with 
this  end  in  view.     (See  section  on  Media.) 

Rothberger  ^  has  devised  a  mixture  of  glucose  agar  to  which  is  added 
one  per  cent  of  a  saturated  aqueous  solution  of  neutral-red.  Shake-cul- 
.tures  or  stab-cultures  are  made  in  tubes  of  this  medium.  The  typhoid 
bacillus  causes  no  changes  in  it,  while  members  of  the  colon  group,  by 
reduction  of  the  neutral-red,  decolorize  the  medium  and  produce  gas  by 
fermentation  of  the  sugar. 

Utilizing  the  fact  that  bile-salts  are  precipitated  in  the  presence  of 
acids,  Macconkey  devised  a  medium  composed  of  sodium  glycocholate, 
pepton,  lactose,  and  agar  (the  composition  of  this  medium  is  given  on 
page  138),  in  which  Bacillus  typhosus  grows  without  causing  nmch 
change,  but  distinct  clouding  results  from  the  growth  of  the  colon  bacillus 
which,  producing  acid  from  the  lactose,  causes  precipitation  of  the  bile- 
salts. 

On  Wurtz's  lactose-litmus-agar  (see  page  129)  the  typhoid  bacillus 
produces  no  acid,  but  eventually  deepens  the  purple  color  to  blue; 
the  colon  bacillus  produces  acid  and  in  stab-cultures  gas  bubbles  and 
the  color  changes  to  red. 

In  Barsiekow's  (see  page  139)  lactose-nutrose-litmus  mixture  the 
typhoid  bacillus  causes  no  change,  while  the  eolon  bacillus  produces 
coagulation  and  an  acid  reaction. 

Cultural  Differences  Within  the  Typhoid  Group. — ^Recent  work  by 
H.  Weiss  in  this  laboratory  has  shown  that  not  all  typhoid  bacilli  are 
culturally  alike,  there  being  two  distinct  groups,  one  which  ferments 
xylose  and  the  other  which  does  not.  This  work  is  being  elaborated. 
Since  there  are  also  antisrenic  differences  it  may  be  necessary  in  the 
future  to  speak  rather  of  a  typhoid  group  than  of  the  typhoid  bacillus. 

Biological  Considerations. — The  typhoid  bacillus  is  an  aerobic  and 
facultatively  anaerobic  organism  growing  well  both  in  the  presence  and  in 
the  absence  of  oxygen  when  certain  sugars  are  present,  showing  a  slight 
preference,  however,  for  well  aerated  conditions.  It  grows  most  luxu- 
riantly at  temperatures  about  37.5°  C,  but  continues  to  grow  within  a 
range  of  temperature  lying  between  15°  and  41°  C.  Its  thermal  death 
point,  according  to  Sternberg,  is  56°  C.  in  ten  minutes.  It  remains  alive 
in  artificial  cultures  for  several  months  or  even  years  if  moisture  is  sup- 
plied.    In  carefully  sealed  agar  tubes  Hiss  has  found   the  organisms 

^Rothberger,  Cent.  f.  Bakt.,  xxiv,  18Q8. 


404  PATHOGENIC  MICROORGANISMS 

alive  after  thirteen  years.  In  natural  waters  it  may  remain  alive  as 
long  as  thirty-six  days,  according  to  Klein/  In  ice,  according  to  Prud- 
den,^  it  may  remain  alive  for  three  months  or  over.  Against  the  ordi- 
nary disinfectants,  the  typhoid  bacillus  is  comparatively  more  resistant 
than  some  other  vegetative  forms.  It  is  killed,  however,  by  1  :  500 
bichlorid  or  five-per-cent  carbolic  acid  within  five  minutes. 

Pathogenicity. — In  animals,  some  early  investigators  to  the  contrary, 
typhoidal  infection  does  not  occur  spontaneously  and  artificial  inocula- 
tion with  the  typhoid  bacillus  does  not  produce  a  disease  analogous  to 
typhoid  fever  in  the  human  being.  Frankel  ^  was  able  to  produce  intes- 
tinal lesions  in  guinea-pigs  by  injection  of  the  bacilli  into  the  duodenum, 
and  recovered  the  bacteria  from  the  spleens  of  the  animals  after  death, 
but  the  disease  produced  was  in  no  other  respect  analogous  to  typhoid 
fever  in  the  human  being.  It  is  probable  that  typhoid  bacilli  injected 
into  animals  do  not  multiply  extensively  and  that  most  of  the  symp- 
toms produced  are  due  to  the  endotoxins  liberated  from  the  dead  bac- 
teria. In  corroboration  of  this  view  is  the  observation  that  inoculation 
with  dead  cultures  is  followed  by  essentially  the  same  train  of  symp- 
toms as  inoculation  with  live  cultures."*  The  injection  of  large  doses  into 
rabbits  or  guinea-pigs  intravenously  or  intraperitoneally  is  usually 
followed  by  a  rapid  drop  in  temperature,  often  by  respiratory  em- 
barrassment and  diarrhea.  Occasionally  blood  may  be  present  in 
the  stools.  According  to  the  size  of  the  dose  or  the  weight  of  the  ani- 
mal, death  may  ensue  within  a  few  hours,  or,  with  progressive  emacia- 
tion, after  a  number  of  days,  or  the  animal  may  gradually  recover. 
Welch  and  Blachstein  ^  have  shown  that  typhoid  baciUi  injected  into 
the  ear  vein  of  a  rabbit  appear  in  the  bile  and  may  persist  in  the  gall- 
bladder for  weeks.  Typhoid  bacilli  isolated  from  different  sources  may 
show  considerable  variations  in  virulence  and  toxicity. 

Doerr,^  Koch,^  Morgan,^  and  more  recently  Johnston^  have  all 
confirmed  this,  the  last  named  showing  that  the  typhoid  bacillus  could 


1  Klein,  Med.  Officers'  Report,  Local  Govern.  Bd.,  London,  1894. 

2  Prudden,  Med.  Rec,  1887. 

3  Frankel,  Cent,  f .  klin.  Med.,  10,  1886. 
*  Petruschky,  Zeit.  f.  Hyg.,  xii,  1892. 

^  Welch  and  Blachstein,  Bull.  Johns  Hop.  Hosp.,  ii,  1891. ' 

8  Doerr,  Centralbl.  f .  Bakt.,  1905. 

'  Koch,  Zeitschr.  f .  Hyg.,  1909. 

^Morgan,  Jour,  of  Hyg.,  1911. 

» Johnston,  Jour,  of  Med.  Res.,  xxvii,  1912.  ' 


BACILLUS  OF  TYPHOID  FEVER  405 

not  only  remain  latent  for  a  long  time  in  the  gall-bladder  of  rabbits, 
but  would  appear  in  the  blood  stream  with  considerable  regularity 
after  the  seventh  or  ninth  day,  and  persist  for  as  long  as  125  days. 
Gay  and  Claypole^  have  been  able  to  produce  the  carrier  state  in 
rabbits  with  great  regularity  by  growing  the  typhoid  cultures  used  for 
inoculation  upon  agar  containing  10  per  cent  defibrinated  rabbit's 
blood.  Such  cultures  are  not  as  readily  agglutinated  by  immune  serum 
as  are  those  grown  on  plain  agar,  and  it  may  well  be  that  they  have 
acquired  a  certain  degree  of  resistance  to  the  serum  antibodies  which 
renders  them  more  competent  to  survive  in  the  body  of  the  rabbit. 
Gay  has  used  rabbits  inoculated  with  such  cultures  for  the  determination 
of  the  efficacy  of  his  sensitized  vaccines. 

In  man  the  large  majority  of  typhoid  infections  take  the  form 
of  the  disease  clinically  known  as  typhoid  fever.  For  a  description 
of  the  clinical  course  and  pathological  lesions  of  the  disease,  the  reader 
is  referred  to  the  standard  text-books  of  medicine  and  pathology. 
During  the  course  of  the  disease,  and  during  convalescence,  the  bacilli 
may  be  cultivated  from  the  circulating  blood,  the  rose  spots,  the  feces, 
the  urine,  and  in  exceptional  cases  from  the  sputum.  At  autopsy  the 
bacilli  may  be  obtained  from  these  sources  as  well  as  from  the  lesions  in 
the  intestine,  the  spleen,  and  often  from  the  liver,  kidneys,  and  from  the 
gall-bladder. 

Though  formerly  regarded  as  primarily  an  intestinal  disease,  recent 
investigations  have  brought  convincing  proof  that  the  disease  is  in  its 
inception  actually  a  bacteriemia.  It  is  not  unlikely  that  the  intestinal 
lesions  are  largely  the  result  of  toxic  products  which  are  excreted 
through  the  intestinal  wall. 

Typhoid  Bacilli  in  the  Blood  during  the  Disease. — The  investigations 
of  many  workers  have  shown  that  typhoid  bacilli  are  present  in  the 
circulating  blood  of  practically  all  patients  during  the  early  weeks  of  the 
disease.  Series  of  cases  have  been  studied  by  Castellani,^  Schottmtil- 
ler,^  and  many  others.  More  recently  Coleman  and  Buxton^  have 
reported  their  researches  upon  123  cases,  and  have  at  the  same  time 
analyzed  all  cases  previously  reported.  Their  analysis  of  blood  cultures 
taken  at  different  stages  in  the  disease  is  as  follows: 


^Gay  and  Claypole,  Arch,  of  Inf.  Med.,  Dec,  1913. 

2  CasteUani,  Riforma  medica,  1900. 

^  Schottmueller,  Deut.  med.  Woch.,  xxxii,  1900,  and  Zeit.  f.  Hyg.,  xxxvi,  1901. 

*  Coleman  and  BuxtoUf  Am.  Jour,  of  Med.  Sci.,  133,  1907. 


406  PATHOGENIC  MICROORGANISMS 

Of  224  cases  during  first  week,  89  per  cent  were  positive. 
Of  484  cases  during  second  week,  73  per  cent  were  positive. 
Of  268  cases  during  third  week,  60  per  cent  were  positive. 
Of  103  cases  during  fourth  week,  38  per  cent  were  positive. 
Of    58  cases  after  fourth  week,  26  per  cent  were  positive. 

The  technique  recommended  by  Coleman  and  Buxton  for  obtaining 
blood  cultures  is  that  recommended  by  Conradi/  slightly  modified.  The 
blood  is  taken  into  flasks  each  containing  about  20  c.c.  of  the  following 
mixture: 

Ox-bile 900  c.c. 

Glycerin 100  c.c. 

Pepton 20  grams. 

About  3  c.c.  of  blood  are  put  into  each  flask.  The  ox-bile,  besides 
preventing  coagulation,  may  possibly  neutralize  the  bactericidal  sub- 
stances present  in  the  drawn  blood.  The  flasks  are  incubated  for  eigh- 
teen to  twenty-four  hours,  at  the  end  of  which  time  streaks  are  made 
upon  plates  of  lactose-litmus-agar  and  the  organisms  identified  by 
agglutination  or  by  cultural  tests. 

European  workers  have  generally  preferred  to  make  high  dilution  of 
the  blood  in  flasks  of  bouillon,  small  quantities  of  blood,  1  to  2  c.c,  being 
mixed  with  100  to  150  c.c.  of  nutrient  broth. 

Epstein  2  has  reported  excellent  results  from  mixing  the  blood  in 
considerable  concentration  with  two-per-cent  glucose  agar  and  pouring 
plates. 

The  writers  in  hospital  work  have  had  equally  good  results  with  the 
bile  medium  and  with  broth  in  flasks,  rather  less  uniform  but  still  satis- 
factory results  with  the  plating  method.  In  general  it  may  be  said  that 
any  one  of  these  methods  carried  out  with  reasonable  accuracy  may  be 
satisfactorily  employed. 

Typhoid  Bacilli  in  the  Stools. — The  examination  of  the  stools  for 
typhoid  bacillus  is  performed  for  diagnostic  purposes  chiefly  in  obscure 
cases.  It  may,  furthermore,  furnish  information  of  great  hygienic 
importance.  Thus  Drigalski  ^  and  Conradi  have  succeeded  in  isolat- 
ing typhoid  bacilli  from  the  stools  of  ambulant  cases  so  mild  that 
they  were  not  clinically  suspected.  It  la  oy  means  of  such  examina- 
tions that  the  so-called  typhoid-carriers  are  detected,   cases  which, 

1  Conradi,  Deut.  med.  Woch.,  xxxii,  1906. 
^Epstein,  Proc.  N.  Y.  Path.  Soc,  N.  S.,  vi,  1906. 
*  Drigalski  and  Conradi,  Zeit.  f .  Hyg.,  xxxix,  1902. 


BACILLUS  OF  TYPHOID  FEVER  407 

though  perfectly  well  themselves,  may  be  a  means  of  spreading  the 
disease.  Such  cases  have  been  known  to  harbor  the  bacilli  for  periods 
as  long  as  several  years. 

The  examination  itself  is  fraught  with  difficulties,  owing  to  the  pre- 
ponderating numbers  of  colon  bacilli  found  in  all  feces  and  the  difficulty 
of  isolating  the  typhoid  bacilli  from  such  mixtures. 

Reviewing  the  data  collected  by  a  number  of  investigators,  it 
seems  probable  that  the  bacilli  do  not  appear  in  the  stools,  at  least 
in  numbers  sufficient  for  recognition,  much  before  the  middle  of  the 
second  week,  or,  in  other  words,  as  pointed  out  by  Hiss,  about  the 
time  that  the  intestinal  lesions  are  well  ad- 
vanced and  ulceration  is  occurring.  Thus 
Wiltschour  ^  could  not  determine  their  pres- 
ence before  the  tenth  day;  Redtenbacher,^ 
in  reviewing  the  statistics,  states  that  in  a 
majority  of  cases  the  bacilH  first  appear 
toward  the  end  of  the  second  week,  and 
Horton-Smith  ^  could  not  find  the  bacilli  be- 
fore the  eleventh  day.  Hiss,^  in  an  investi- 
gation of  the  same  subject,  obtained  the 
following  results: 

First  to  tenth  day,  inclusive,  twenty-         fiq.  89.— Bacillus  coli. 
eight  cases  examined;  typhoid  bacilli  isolated     Deep  colonies  on  Hiss  plate 
from  three;   percentage   of  positive    cases     medium. 
10.7  per  cent. 

Eleventh  to  twentieth  day,  inclusive,  forty-four  cases  examined;  ty- 
phoid bacilli  from  twenty-two;  percentage  of  positive  cases  50  per  cent. 

Twenty-first  day  to  convalescence,  sixteen  cases  examined;  typhoid- 
bacilli  isolated  from  thirteen;  percentage  of  positive  cases  81.2  per 
cent. 

The  difficulties  encountered  in  such  examinations  have  led  to  the 
development  of  a  large  number  of  methods.  The  first  method  which 
yielded  successful  results  was  that  of  Eisner,^  who  employed  a  potato- 
extract  gelatin  containing  one  per  cent  of  potassium  iodid,  a  medium 
which  prevented  the  growth  of  many  intestinal  bacteria,  allowing  only 

1  Wiltschour,  Cent.  f.  Bakt.,  1890.  — 

2  Redtenbacher,  Zeit.  f .  klin.  Med.,  xix,  1891. 

*  Horton-Smith,  Lancet,  May,  1899. 

*  Hiss,  Med.  News,  May,  1901. 

6  Eisner,  Zeit.  f .  Hyg.,  xxi,  1895. 


0 

1 

o 

Of 

^ 

■%«' 

■  0 

408 


PATHOGENIC  MICROORGANISMS 


colon,  typhoid,  and  a  few  others  to  develop.    This  medium  is  at  present 
rarely  used. 

Hiss  ^  has  employed  with  success  an  agar-gelatin  mixture  containing 
one  per  cent  of  glucose,  the  preparation  of  which  has  been  described  in 
detail  in  the  section  on  media.  The  actual  technique  of  the  test  is  as 
follows:  One  to  two  loopfuls  of  feces  are  transferred  to  a  tube  of  broth, 
making  the  broth  fairly  cloudy.  From  this  emulsion  five  or  six  plates 
are  made  by  transferring  in  series  one  to  five  loopfuls  of  the  emulsion 
to  tubes  containing  the  melted  plate  medium,  and  then  pouring  the  con- 
tents of  these  tubes  into  Petri  dishes.     These  dishes,  after  the  medium 


Fig.  90. — ^Bacillus  typhosus.    Deep  colonies  in  Hiss  plate  medium. 


has  hardened,  are  placed  in  an  incubator  at  37°  C,  and  allowed  to  re- 
main for  eighteen  to  twenty-four  hours,  when  they  are  ready  for  examina- 
tion. If  typhoid  bacilli  are  present  they  will  be  found  as  small,  usually 
glistening  colonies  with  a  fringe  of  threads  growing  out  like  flagella  from 
their  peripheries  (see  Figs.  90  and  91).  These  colonies  are  smaller  and 
quite  distinct  from  those  of  colon  bacilU,  which  are  heavier  and  darker 
and  do  not  display  the  fringing  threads.  Suspicious  colonies  may  be 
fished  and  transferred  to  the  Hiss  tube  medium  (see  page  133)  or  iden- 
tified by  other  reliable  methods. 

A  method  which  has  been  found  useful,  especially  in  Europe, 
is  that  in  which  smears  of  diluted  feces  are  made  upon  large  plates 
of  the  Conradi-Drigalski  medium.  (For  preparation  see  page  135.) 
The  principles  underlying  the  use  of  this  medium  are  the  formation 
of  acid  from  the  lactose  by  the  colon  bacilli  and  the  inhibition  of  cocci 
and  many  other  bacteria  by  the  crystal-violet.     In  practice,  an  emul- 

1  Hiss,  Jour,  of  Exp,  Med.,  ii,  1897;  Med.  News,  May,  1901;  and  Jour.  Med.  Res., 
N.  S.,  iii,  1902. 


BACILLUS  OF  TYPHOID  FEVER  409 

sion  is  made  of  a  loopful  of  feces  in  a  tube  of  broth.  Into  this 
is  dipped  a  bent  glass  smearing  rod,  the  excess  of  fluid  is  allowed  to 
drip  off,  and  smears  are  made  upon  plates  of  the  medium,  several 
plates  being  smeared  without  redipping  the  rod.  Colonies  of  the 
colon  bacillus  on  these  plates  will  appear  opaque,  comparatively  large, 
and  will  produce  an  acid  reaction  with  consequent  reddening  of  the 
medium.  Typhoid  colonies  will  be  smaller,  transparent,  and  without 
acid  formation.    These  colonies  are  fished  and  the  microorganisms  may 


Fig.  91. — Bacillus  typhosus.    Colony  in  Hiss  plate  medium,  highly  magnified. 

be  identified  by  agglutination  or  by  stab  cultures  in  the  Hiss  tube 
medium. 

The  malachite-green  media  of  Loeffler  and  others  have  found 
less  general  use  than  was  originally  expected,  because  of  the 
difficulty  in  obtaining  uniform  preparations  of  malachite-green. 
Peabody  and  Pratt  ^  have  applied  the  principle  of  colon-bacillus  in- 
hibition by  malachite-green,  by  adding  this  dye  to  broth  in  the 
manner  described  in  the  section  on  media  (page  137),  planting  the 
feces  directly  into  this  broth,  and,  after  incubation  for  several  hours, 
making  smears  from  these  tubes  upon  plates  of  the  Conradi-Drigalski 
medium. 
I      As  a  routine  method  for  isolation  of  the  bacilli  from  stools  we  our- 

1  Peabody  and  Pratt,  Boston  Med.  and  Surg.  Jour.,  1908. 
27 


410 


PATHOGENIC  MICROORGANISMS 


selves  use  largely  the  Endo  fuchsin-agar  or  Kendall's  modified  Endo 
medium.  (See  p.  135.)  Emulsions  of  feces  are  made  in  tubes  of 
ordinary  broth  or  salt  solution  and  smears  of  this  emulsion  are  made 
upon  several  large  plates  of  the  fuchsin-agar  by  means  of  a  glass  smearing 
rod.  The  colonies  of  Bacillus  coli,  after  eighteen  or  more  hours  of 
incubation,  will  be  found  to  have  brought  back  a  deep  red  color  to  the 
medium,  whereas  the  typhoid  colonies  are  small,  more  transparent,  and 
have  left  the  medium  uncolored. 

An  excellent  medium  recently  devised  is  that  of  Krumwiede.  (See 
p.  136.) 

In  all  cases  where  plates  are  prepared  from  broth  emulsions  of  feces, 
it  is  desirable  t9  allow  the  emulsion  to  stand  at  incubator  temperature 


Fig.  92. — Colon  and  Typhoid  Colonies  in  Hiss  Plate  Medium. 
from  stool.     Note  the  small  thread-forming  typhoid  colonies.) 


(Planted 


for  an  hour.  Subsequent  removal  of  fluid  from  the  upper  layers  of  the 
medium  is  likely  to  bring  away  a  comparatively  larger  number  of  the 
organisms. 

The  methods  given  above  do  not  exhaust  the  records  of  work  done 
upon  this  problem.  It  is  not  satisfactory  to  compare  any  two  methods 
as  to  practical  value,  since  all  of  them  require  famiharity  with  organisms 
and  media.  In  fact,  it  may  be  said  that  all  of  the  methods  given  are 
satisfactory  if  consistently  employed  by  a  worker  who  has  become 
thoroughly  accustomed  to  the  peculiarities  of  the  typhoid  colonies  upon 
the  medium  with  which  he  is  working. 

In  all  of  these  methods  when  suspicious  colonies  are  found  they  are 
identified  morphologically  and  transplanted  to  such  media  as  the  Russell 
double-sugar  agar,  or  the  Hiss  tube  medium.     For  rapid  diagnosis 


BACILLUS  OF  TYPHOID  FEVER  411 

agglutination  may  be  done  in  a  strong  immune  serum,  either  directly 
from  the  colony  by  the  hang-drop  method,  or  better  macroscopically 
from  the  growth  in  the  transplant. 

Typhoid  Bacilli  in  the  Urine. — Careful  investigation  has  revealed 
typhoid  bacilli  in  the  urine  in  about  twenty-five  per  cent  of  all  patients. 
Neimiann  ^  discovered  the  bacilli  in  eleven  out  of  forty-six  and  Kar- 
linsky  ^  in  twenty-one  out  of  forty-four  cases.  Investigations  by  Pe- 
truschy,^  Richardson,^  Horton-Smith,^  Hiss,^  and  others  have  confirmed 
these  results.  In  general  the  bacilli  have  not  been  found  before  the 
fifteenth  day  of  the  disease,  and  examination  of  the  urine,  therefore, 
can  be  of  little  early  diagnostic  value.  A  series  of  seventy-five  cases 
examined  by  Hiss  before  the  fourteenth  day  of  the  disease  did  not  once 
reveal  typhoid  bacilli  in  the  urine.  On  the  other  hand,  they  have  been 
found  to  be  present  for  weeks,  months,  and,  in  isolated  cases,  for  years 
after  convalescence,  the  examination  thus  having  much  hygienic  im- 
portance. They  are  probably  present  in  about  twelve  per  cent  of  cases 
during  the  early  days  of  convalescence.  In  most  of  these,  albumin  is 
present  in  the  urine  in  considerable  quantities.  The  bacilli  usually 
appear  and  disappear  with  the  albuminuria. 

\  An  obstinate  cystitis  caused  by  typhoid  bacilli  may  follow  in  the 
path  of  typhoid  fever.  Such  cases  have  been  reported  by  Blumer,^ 
Richardson,^  and  others.  Suppurative  processes  in  the  kidneys  are 
less  frequent.  It  is  noteworthy,  also,  that  in  the  course  of,  and  fol- 
lowing, typhoid  fever  there  often  occurs  voiding  of  Bacillus  coli  with 
the  urine.  This  may  obstinately  persist  for  considerable  periods  after 
convalescence.    The  reasons  for  this  are  not  entirely  clear. 

Typhoid  Carriers  and  Typhoid  Bacilli  in  the  Gall-Bladder. — Typhoid 
bacilli  have  been  frequently  observed  in  the  gall-bladder  at  autopsy. 
They  have  also  been  found  present  in  this  organ,  at  operations  for 
cholecystitis,  months  and  years  after  the  occurrence  of  typhoid  fever. 
Miller  *  has  reported  a  case  in  which  typhoid  bacilli  were  present  in  the 
gall-bladder  seven  years  after  the  disease;  v.  Dungern^^  has  cultivated 
them  from  an  inflamed  gall-bladder  fifteen  years  after  the  disease. 
Zinsser  has  had  occasion^^  to  observe  a  case  in  which  an  operation  for 

1  Neumann,  Berl.  klin.  Woch.,  xxvii,  1890.     «  Hiss,  Med.  News,  May,  1901. 

2  Karlinsky,  Prag.  med.  Woch.,  xv,  1890.  "^  Blunter,  Johns  Hopk.  Hosp.  Rep.,  5,  1895. 
'  Petruschy,  Cent.  f.  Hyg.,  xxiii,  1898.  ^  Richardson,  loc.  cit. 

^  Richardson,  Jour.  Exp.  Med.,  3, 1898.  ^  Miller,  Johns  Hopk.  Hosp.  Bull.,  1898. 

^  Horton-Smith,  Lancet,  May,  1899.  "  v.  Dungern,  Miinch.  med.  Woch.,  1897. 

»  Zimser,  Proc.  N.  Y.  Pathol.  Soc,  1908. 


412  PATHOGENIC  MICROORGANISMS 

gall-stone  seventeen  years  after  the  occurrence  of  typhoid  fever  revealed 
the  presence  of  the  bacilli  in  the  gall-bladder.  In  such  cases  typhoid 
bacilli  may  be  constantly  discharged  from  the  intestine  with  the  feces 
and  prove  a  menace  to  the  health  of  the  community.  An  extremely  in- 
teresting example  of  such  a  typhoid  carrier  has  been  carefully  studied 
and  reported  by  Park.^ 

Typhoid  carriers  are  much  more  common  than  formerly  supposed. 
Lentz  2  believes  that  about  4  per  cent  of  all  cases  become  chronic  car- 
riers and  Goldberger,  averaging  the  cases  of  other  workers,  calculates 
that  of  1,782  cases  of  typhoid,  53  or  about  3  per  cent  became  chronic 
carriers.  This  problem  therefore  is  of  the  utmost  sanitary  import- 
ance. Even  when  detected  the  cure  of  such  carriers  is  very  difficult, 
and  perhaps,  in  some  cases  impossible  without  cholecystectomy.  Vac- 
cine treatment  has  not  been  encouraging  and  no  other  form  of  uni- 
formly successful  treatment  has  so  far  been  devised. 

Typhoid  Bacilli  in  the  Rose  Spots. — Neufeld  ^  obtained  positive  re- 
sults in  thirteen  out  of  fourteen  cases.  According  to  his  researches  and 
those  of  Frankel,^  the  bacilli  are  localized  not  in  the  blood,  which  is 
taken  when  the  rose  spots  are  incised,  but  are  crowded  in  large  num- 
bers within  the  lymph  spaces. 

Typhoid  Bacilli  in  the  Sputum. — In  rare  cases  typhoid  bacilli  have 
been  found  in  the  sputum  of  cases  complicated  by  bronchitis,  broncho- 
pneumonia, and  pleurisy.  Such  cases  have  been  reported  by  Chante- 
messe  and  Widal,^  Frankel,®  and  a  number  of  others.  Empyema,  when 
it  occurs  in  connection  with  such  cases,  is  usually  accompanied  by  a 
mixed  infection.  From  a  hygienic  point  of  view  the  spread  of  typhoid 
fever  by  means  of  the  sputum  is  probably  of  rare  occurrence. 

Suppurative  Lesions  Due  to  Typhoid  Bacillus. — In  the  course  of 
typhoid  convalescence  or  during  the  latter  weeks  of  the  disease,  sup- 
purative lesions  may  occur  in  various  parts  of  the  body.  The  most 
frequent  localization  of  these  is  in  the  periosteimi,  especially  of  the  long 
bones,  and  in  the  joints.  A  large  number  of  such  lesions  have  been 
described  by  Welch,  Richardson,^  and  others.  They  usually  take  the 
form  of  periosteal  abscesses,  often  located  upon  the  tibia,  occurring 

^Park,  "Pathogenic  Bacteria,"  N.  Y.,  1908. 

2  Lentz,  Hyg.  Rundscha,u,  vol.  16,  1906. 

'  Neufeld,  Zeit.  f.  Hyg.,  xxx,  1899.  *  Frankel,  Zeit.  f.  Hyg.,  xxxiv,  1900. 

^  Chantemesse  and  Widal,  Arch,  de  physiol.  norm,  et  path.,  1887. 

*  Frankel,  Deut.  med.  Woch.,  xv  and  xvi,  1899. 

'  Richardson,  Jour.  Boston  Soc.  Med.  Sci.,  5,  1900. 


BACILLUS  OF  TYPHOID  FEVER  413 

either  late  in  the  disease  or  months  after  convalescence,  and  are  char- 
acterized by  very  severe  pain.  Osteomyelitis  may  also  occur,  but  is 
comparatively  rare.  Subcutaneous  abscesses  and  deep  abscesses  in  the 
muscles,  due  to  this  bacillus,  have  been  described  by  Pratt.  ^  Synovitis 
may  also  occur. 

Meningitis,  due  to  the  typhoid  bacillus,  occurs  not  infrequently, 
usually  during  convalescence  from  typhoid  fever.  A  case  of  primary 
typhoid  meningitis  has  been  reported  by  Farnet.^ 

Peritoneal  abscesses,  due  to  the  typhoid  bacillus,  have  been  re- 
ported. The  writer  ^  has  reported  a  case  in  which  typhoid  bacilli  were 
found  free  in  the  peritoneal  cavity  during  typhoid  fever  without  per- 
foration of  the  gut. 

Isolated  instances  of  typhoid  bacilli  in  abscesses  of  the  thyroid  and 
parotid  glands  and  in  brain  abscesses  have  been  observed. 

Typhoid  Fever  without  Intestinal  Lesions. — A  considerable  number 
of  cases  have  been  reported  in  which  typhoid  bacilli  have  been  isolated 
from  the  organs  after  death  or  from  the  secretions  during  life  of  pa- 
tients in  whom  the  characteristic  lesions  of  typhoid  fever  have  been 
backing.  Most  of  these  cases  must  be  regarded  as  true  typhoid  septi- 
cemias. In  some  cases  the  bacilli  were  isolated  from  the  spleen,  liver^ 
or  kidneys;  in  others,  from  the  urine  or  the  gall-bladder.  In  a  case 
observed  by  Zinsser  the  bacilli  were  isolated  from  an  infarct  of  the 
kidney  removed  by  operation.  In  this  case  the  clinical  course  of  the 
disease  had  pointed  only  toward  the  existence  of  an  indefinite  fever  ac- 
companied by  symptoms  referable  to  the  kidneys.  The  Widal  test, 
however,  was  positive.  An  excellent  summary  of  such  cases,  together 
with  several  personally  observed,  has  been  given  by  Flexner."^ 

Hygienic  Considerations. — Although  typhoid  fever  is  frequently 
spoken  of  as  an  epidemic  disease,  it  is,  more  truly,  endemic  in  character 
in  almost  all  parts  of  the  world,  but  subject  to  occasional  epidemic  ex-, 
acerbations.  i  In  the  larger  communities  of  the  temperate  zones  these 
epidemics  take  place  chiefly  in  the  autumn  and  are  circumscribed  by 
the  distribution  of  a  water  or  milk  supply. 

Since  the  disease  never  occurs  except  by  transmission,  directly  or  in- 
directly from  a  previous  case,  it  is  amenable  more  than  most  other  mal- 
adies to  sanitary  regulation,  and  it  may  be  said  that  extensive  preva- 
lence of  typhoid  in  a  large  community  is  a  consequence  of  defect  in  the 

1  Pratt,  Jour.  Boston  Soc.  Med.  Sci.,  3,  1899. 

2  Famet,  Bull,  de  la  soc.  med.  des  hop.  de  P.,  3,  1891. 

»  Zinsser,  Proc.  N.  Y.  Path.  Soc,  1907.        *  Flexner,  Johns  Hopk.  Rep.,  5,  1896. 


414  PATHOGENIC  MICROORGANISMS 

system  of  sanitation.    The  disease  is  acquired  by  ingestion  of  the  spe-       ^ 
cifie  bacteria.    Infection  by  other  channels  has  not  been  demonstrated. 

Prophylactic  measures  in  typhoid  fever,  therefore,  should  begin 
with  the  isolation  of  the  patient  and  the  disinfection  of  excreta,  dis- 
charges, linen,  and  all  utensils  which  have  been  in  contact  with  the 
patient.  The  bacilli  leave  the  body  in  the  feces  and  the  urine  and  the 
dangers  of  contaminatiori,  by  these  substances,  of  all  objects  in  imme- 
diate contact  with  the  patient  are  considerable.  Excreta  should  there- 
fore be  either  mixed  with  boiling  water  or  chemically  disinfected,  pref- 
erably by  means  of  thoroughly  mixing  with  carbolic  acid,  lysol,  or  a  solu- 
tion of  freshly  slaked  lime,  and,  if  possible,  destroyed  by  burning. 
Linen,  tableware,  and  eating  utensils  should  be  soaked  in  similar  solu- 
tions and  boiled.  The  observance  of  such  measures,  furthermore, 
should  not  be  discontinued  until  bacteriological  examination  has  demon- 
strated the  absence  of  the  bacilli  from  feces  and  urine.  Disregard  of 
this  last  precaution  may  well  be  one  of  the  main  causes  of  the  endemic 
persistence  of  the  disease  in  large  cities — especially  considered  in  the 
light  of  our  recent  knowledge  of  'Hyphoid  carriers." 

Typhoid  fever,  in  the  large  majority  of  cases,  is  transmitted  by 
the  agency  of  water.  In  an  analysis  of  six  hundred  and  fifty  tjrphoid 
epidemics  Schiider  ^  found  four  hundred  and  sixty-two  reported,  upon 
reasonable  evidence,  as  originating  from  water.  The  technical  difficul- 
ties attending  the  isolation  of  typhoid  bacilli  from  contaminated  water 
have  prevented  actual  bacteriological  proof  in  most  epidemics;  never- 
theless, indirect  evidence  of  pollution  of  the  suspected  water-supply, 
correspondence  of  the  distribution  of  this  supply  with  that  of  the  dis- 
ease, and  reduction  of  typhoid  morbidity  upon  the  substitution  of  an 
uncontaminated  supply  are  sufficiently  convincing  to  remove  reasonable 
doubt.  Added  to  this  is  our  knowledge,  from  the  experiments  of  Jor- 
dan, Russell,  and  Zeit  ^  and  others,  that  typhoid  bacilli  may  remain 
alive  in  natural  waters  for  as  long  as  five  days.  Prudden  has  demon- 
strated that  the  bacilli  may  survive  freezing  as  long  as  three  months. 

Next  to  water,  the  most  important  source  of  typhoid  fever  is  con- 
taminated milk.  In  the  summary  by  Schiider,^  one  hundred  and  ten 
of  the  four  hundred  and  sixty  epidemics  were  attributable  to  milk. 
No  visible  modifications  in  milk  occur,  which  makes  this  source  es- 
pecially insidious.  Contamination  of  milk  has  been  traceable  to  water 
used  in  washing  cans  or  to  dairy  attendants. 

*  Schiider,  Zeit.  f.  Hyg.,  xxxviii,  1901. 

2  Jordan,  Russell,  and  Zeit,  Jour,  of  Inf.  Dis.,  1,  1904.  ^  Schiider,  loc.  cit. 


BACILLUS  OF  TYPHOID  FEVER  415 

Excluding  water  and  milk,  all  remaining  causes  of  typhoid  dissemi- 
nation constitute  about  twelve  per  cent  and  are  found  chiefly  in  the 
use  of  vegetables  contaminated  from  infected  soil,  and  other  food  prod- 
ucts. Recently  Conn  ^  suggested  that  oysters  grown  in  waters  close 
to  sewage  discharges  may  be  the  means  of  typhoid  transmission.  An 
epidemic  at  Wesleyan  University  was  traced  to  this  cause.  Experi- 
ments by  Foote  ^  demonstrated  that  typhoid  bacilli  may  be  found 
alive  within  oysters  for  three  weeks  after  they  disappeared  from  the 
surrounding  water.  The  importance  of  this  mode  of  infection  is  un- 
certain. Rosenau,  Lumsden,  and  Castle  ^  found  it  to  be  negligible  in 
the  District  of  Columbia. 

Indirect  contamination  of  food  and  water  by  the  intermediation  of 
flies  and  other  insects  has  been  emphasized  by  Veeder  *  and  is  unques- 
tionably of  great  importance. 

Typhoid  Carriers. — One  of  the  important  factors  in  the  spread 
of  typhoid  fever  is  the  existence  of  a  considerable  number  of  ''car- 
riers ' '  in  every  community.  These  are  individuals  who  harbor  typhoid 
bacilli  in  their  intestinal  canals,  probably  with  a  nidus  in  the  gall 
bladder.  They  may  intermittently  or  continually  discharge  typhoid 
bacilli  with  the  feces  and  therefore  be  a  constant  menace  to  others. 
There  are  now  a  considerable  number  of  well-known  cases  where  series 
of  small  epidemics  have  been  started  by  carriers,  and  in  the  mobiliza- 
tion of  armies  the  incorporation  of  carriers  with  troops  may  be  a  seri- 
ous menace. 

Carriers  may  be  discovered  by  isolation  of  the  bacilli  from  the 
feces  as  described  in  another  place. 

Poisons  of  the  Tsrphoid  Bacillus. — The  investigation  of  the  toxic 
products  of  the  typhoid  bacillus  has  occupied  the  attention  of  a  large 
number  of  workers.  The  first  to  do  experimental  work  upon  the  sub- 
ject was  Brieger  ^  soon  after  the  discovery  and  cultivation  of  the  micro- 
organism. That  toxic  substances  can  be  obtained  from  typhoid  cultures 
is  beyond  question.  There  is,  however,  a  definite  difference  of  opinion 
as  to  whether  these  poisons  are  so-called  endotoxins  only,  or  whether 
they  are  in  part  composed  of  soluble  toxins  comparable  to  those  of 
diphtheria  and  tetanus,  follovnng  the  injection  of  which  antitoxic  sub- 
stances may  be  formed. 

The  evidence  so  far  seems  to  bear  out  the  original  contention  of 

1  Conn,  Med.  Record,  Dec,  1894.  2  poote,  Med.  News,  1895. 

'  Rosenau,  Lumsden,  and  Castle,  Bull.  52,  Hyg.  Lab.  U.  S.  Pub.  Health  Service,  1908. 

*  Veeder,  Med.  Record,  45,  1898.  ^  Brieger,  Deut.  med.  Woch.,  xxvii,  1902. 


416  PATHOGENIC  MICROORGANISMS 

Pfeiffer/  who  first  advanced  the  opinion  that  the  poisonous  substances 
are  products  of  the  bacterial  body  set  free  by  destruction  of  the  bacteria 
by  the  lytic  substances  of  the  invaded  animal  or  human  being.  These 
poisons,  when  injected  into  animals  for  purposes  of  immunization,  in 
Pfeiffer's  experiments,  did  not  incite  the  production  of  neutralizing  or 
antitoxic  bodies,  but  of  bactericidal  and  lytic  substances.  That  these 
endotoxins  constitute  by  far  the  greater  part  of  the  toxic  products  of 
the  typhoid  bacillus  can  be  easily  demonstrated  in  the  laboratory,  by 
the  simple  experiment  of  filtering  a  young  typhoid  culture  (eight  or 
nine  days  old)  and  injecting  into  separate  animals  the  residue  of  bacilli 
and  the  clear  filtrate  respectively.  In  such  an  experiment  there  will  be 
little  question  as  to  the  overwhelmingly  greater  toxicity  of  the  bacillary 
bodies  as  compared  with  that  of  the  culture  filtrate.  On  the  other 
hand,  if  such  cultures,  especially  in  alkaline  media,  are  allowed  to 
stand  for  several  months  and  the  bacilh  thus  thoroughly  extracted  by 
the  broth,  the  toxicity  of  the  filtrate  is  found  to  be  greatly  increased. 

Nevertheless,  more  recent  experiments  by  Besredka,^  Macfadyen,^ 
Kraus  and  Stenitzer,*  and  others  have  tended  to  show  that,  together 
with  such  endotoxic  substances,  typhoid  bacilli  may  produce  a  true 
toxin  which  is  not  only  obtainable  by  proper  methods  from  compara- 
tively young  typhoid  cultures,  but  which  fulfils  the  necessary  require- 
ment of  this  class  of  poisons  by  producing  in  treated  animals  a  true 
antitoxic  neutralizing  body. 

The  typhoid  endotoxins  may  be  obtained  by  a  variety  of  methods. 
Hahn^  has  obtained  what  he  calls  "  typhoplasmin '^  by  subjecting  them 
to  a  pressure  of  about  four  hundred  atmospheres  in  a  Buchner  press. 
The  cell  juices  so  obtained  are  cleared  by  filtration.  Macfadyen  has 
obtained  typhoid  endotoxins  by  triturating  the  bacilli  after  freezing 
them  with  liquid  air  and  extracting  in  1  :  1,000  potassium  hydrate. 
Besredka  obtained  toxic  substances  by  emulsifying  agar  cultures  of 
bacilli  in  salt  solution,  sterilizing  them  by  heating  to  60°  C.  for  about 
one  hour,  and  drying  in  vacuo.  The  dried  bacillary  mass  was  then 
ground  in  a  mortar  and  washed  in  sterile  salt  solution  which  was 
again  heated  to  60°  C.  for  two  hours.    The  remnants  of  the  bacterial 

1  Pfeiffer,  Deut.  med.Woch.,  xlviii,  1894;  Pfeiffer  und  Kolle,  Zeit.  f.  Hyg.,  xxi,  1896. 

2  Besredka,  Ann.  de  Tinst.  Pasteur,  1895,  1896. 

^Macfadyen  and  Rowland,  Cent.  f.  Bakt.,  I,  xxx,  1901;    Macfadyen,   Cent,  f. 
Bakt.,  I,  1906. 

*  Kraus  und  Stenitzer,  Quoted  from  "Handb.d.  Tech., "etc.,  1,  Fischer,  Jena,  1907. 
« Hahn,  Miinch.  med.  Woch.,  xxiii,  1906. 


C:. 


BACILLUS  OF  TYPHOID  FEVER  ^17 

bodies  settle  out  and  the  slightly  turbid  supernatant  fluid  contains  the 
toxic  substances. 

Vaughan  ^  has  obtained  poisons  from  typhoid  bacilli  by  extracting 
at  78°  C.  with  a  two-per-cent  solution  of  sodium  hydrate  in  absolute 
alcohol.  In  this  way  he  claims  to  separate  by  hydrolysis  a  poisonous 
and  a  non-poisonous  fraction.  He  claims,  moreover,  that  this  poison- 
ous fraction  is  similar  to  the  poisons  obtained  in  the  same  way  from 
Bacillus  coli  and  the  tubercle  bacillus,  and  other  proteid  substances, 
beheving  that  the  specific  nature  of  such  proteids  depends  upon  the 
non-toxic  fraction. 

A  simple  method  of  obtaining  toxins  from  typhoid  bacilli  is  carried 
out  by  cultivating  the  microorganisms  in  meat-infusion  broth,  rendered 
alkaline  with  sodium  hydrate  to  the  extent  of  about  one  per  cent. 
The  cultures  are  allowed  to  grow  for  two  or  three  weeks  and  then  steril- 
ized by  heating  to  60°  C.  for  one  hour,  and  allowed  to  stand  for  three 
or  four  weeks  at  room  temperature.  At  the  end  of  this  time  the  cul- 
tures may  be  filtered  through  a  Berkefeld  or  Pasteur-Chamberland  filter 
and  will  be  found  to  contain  strong  toxic  substances. 

The  accounts  concerning  the  thermostability  of  the  various  toxins 
obtained  are  considerably  at  variance.  In  general,  corresponding  with 
other  endotoxins,  observers  agree  in  considering  them  moderatiely  re- 
sistant to  heat,  rarely  being  destroyed  at  temperatures  below  70°  C. 

Intravenous  inoculation  of  rabbits  with  typhoid  endotoxins,  if  in 
sufficient  quantity,  produces,  usually  within  a  few  hours,  a  very  marked 
drop  in  temperature,  diarrhea,  respiratory  embarrassment,  and  death. 
If  given  in  smaller  doses  or  by  other  methods  of  inoculation — ■ 
subcutaneous  or  intraperitoneal — rabbits  are  rendered  extremely  ill, 
with  a  primary  drop  in  temperature,  but  may  live  for  a  week  or  ten  dayS, 
and  die  with  marked  progressive  emaciation,  or  may  survive.  Guinea- 
pigs  and  mice  are  susceptible  to  the  endotoxins,  though  somewhat 
less  so  than  rabbits.  • 

Immunity  in  Typhoid  Fever. — As  a  rule,  one  attack  of  typhoid  fever 
protects  against  subsequent  ones.  Although  exceptions  to  this  rule 
may  occur,  they  are  so  rare  that  the  history  of  a  previous  attack  of  this 
disease  practically  excludes  its  consideration  in  the  diagnosis  of  any 
obscure  condition. 

Animals  may  be  actively  immunized  by  the  injection  of  typhoid 
bacilli  in  gradually  increasing  doses.     In  actual  practice,  this  is  best 

1  Vaughan,  Am.  Jour,  of  Med.  Sci.,  136,  No.  3,  1908. 


418  PATHOGENIC  MICROORGANISMS 

accomplished  by  beginning  with  an  injection  of  about  1  c.C.  of  broth 
culture  heated  for  ten  minutes  at  60°  in  order  to  kill  the  bacilli.  After 
five  or  six  days,  a  second  injection  of  a  larger  dose  of  dead  bacilli  is 
administered;  at  similar  intervals,  gradually  increasing  doses  of  dead 
bacilli  are  given  and  finally  considerable  quantities  of  a  living  and  full)'' 
virulent  culture  may  be  injected  without  serious  consequences  to  the 
animal.  While  this  method  is  convenient  and  usually  successful,  it 
is  also  possible  to  obtain  satisfactory  immunization  by  beginning  with 
very  small  doses  of  living  microorganisms,  according  to  the  early 
method  of  Chantemesse  and  Widal,^  and  others. 

Such  active  immunization,  successfully  carried  out  upon  rabbits  and 
guinea-pigs,  within  a  short  time  after  the  discovery  of  the  typhoid  bacil- 
lus, was  believed  to  depend  upon  the  development  of  antitoxic  sub- 
stances in  immunized  animals.  This  point  of  view,  however,  was  not 
long  tenable,  and  was  definitely  disproven  by  the  investigations  of  Pfeif- 
fer  and  KoUe  ^  in  1896.  These  investigators,  as  well  as  a  large  number  of 
others  working  subsequently,  have  shown  satisfactorily  that  there  are 
present  in  the  blood  serum  of  typhoid-immune  animals  and  human 
beings,  bacteriolytic,  bactericidal,  and  agglutinating  substances,  and  to 
a  lesser  extent,  precipitating  and  opsonic  bodies. 

Bactericidal  and  Bacieriolytic  Substances. — The  bacteriolytic  sub- 
stances in  typhoid-immune  serum  may  be  demonstrated  either  by  the 
intraperitoneal  tachnique  of  Pfeiffer  or  in  vitro.  In  the  former  experi- 
ment a  small  quantity  of  a  fresh  culture  of  typhoid  bacilli  is  mixed 
with  the  diluted  immune  serum  and  the  emulsion  injected  into  the 
peritoneal  cavity  of  a  guinea-pig.  Removal  of  peritoneal  exudate  with 
a  capillary  pipette  and  examination  in  the  hanging  drop  will  reveal, 
within  a  short  time,  a  swelling  and  granulation  of  the  bacteria — the 
so-called  Pfeiffer  phenomenon.  The  test  in  vitro,  as  recommended  by 
Stern  and  Korte,^  may  be  carried  out  by  adding  definite  quantities  of 
a  fresh  agar  culture  of  typhoid  bacilli  to  progres^vely  increasing  dilu- 
tions of  inactivated  immune  serum  together  with  definite  quantities  of 
complement  in  the  form  of  fresh  normal  rabbit  or  guinea-pig  serum. 
At  the  end  of  several  hours'  incubation  at  37.5°  C.  definite  quantities 
of  the  fluid  from  the  various  tubes  are  inoculated  into  melted  agar 
and  plates  are  poured  to  determine  the  bactericidal  action.  Careful 
colony  counting  in  these  plates  and  comparison  with  proper  controls 

'  Chantemesse  and  Widal,  Ann,  de  llnst.  Pasteur,  1892. 

2  Pfeiffer  und  Kolle,  Zeit.  f.  Hyg.,  xxi,  1896. 

3  Stern  und  Korte,  Berl.  klin.  Woch.,  x.,  1904. 


BACILLUS  OF  TYPHOID  FEVER  419 

will  not  only  definitely  demonstrate  the  presence  of  bactericidal  sub- 
stances in  the  immune  serum,  but  will  furnish  a  reasonably  accurate 
quantitative  estimation.     (For  these  tests  see  p.  255.) 

Although  normal  human  serum  contains  in  small  quantity  substances 
bactericidal  to  typhoid  bacilH,  moderate  dilution,  1  :  10  or  1  :  20,  of  such 
sei*um  will  usually  suffice  to  eliminate  any  appreciable  bactericidal  action. 
The  bactericidal  powers  of  immune  serum,  on  the  other  hand,  are  often 
active,  according  to  Stem  and  Korte,  in  dilutions  of  over  1  :  4,000  and  in 
one  case  even  of  1  :  4,000,000.  The  specificity  of  such  reactions  gives 
them  a  considerable  degree  of  practical  value,  both  in  the  biological 
identification  of  a  suspected  typhoid  bacillus  in  known  serum  and  in  the 
diagnosis  of  typhoid  fever  in  the  human  patient  by  the  action  of  the 
patient's  serum  on  known  typhoid  bacilli.  In  the  publication  of  Stem 
and  Korte,  quoted  above,  it  was  found  that  typhoid  patients  during  the 
second  week  often"  possess  a  bactericidal  power  exceeding  1  :  1,000, 
whereas  the  blood  of  normal  human  beings  was  rarely  active  in  dilu- 
tions exceeding  1  :  50  or  1  :  100.  While  scientifically  accurate,  the  prac- 
tical application  of  bactericidal  determinations  for  diagnosis  presents 
considerable  technical  difficulties,  and  gives  way  to  the  no  less  accurate 
method  of  agglutination. 

Agglutinins. — Agglutinins  are  formed  in  animals  and  man  inoculated 
with  typhoid  bacilli,  and  in  the  course  of  typhoid  fever.  It  was,  in  fact, 
while  studying  the  typhoid  bacillus  that  the  agglutinins  were  first  dis- 
covered by  Gruber  and  Durham. 

In  animals,  by  careful  immunization,  specific  typhoid  agglutinins 
may  easily  be  produced  in  sufficient  quantity  to  be  active  in  dilution 
of  1  :  10,000,  and  occasionally  even  1  :  50,000  or  over.  In  the  blood 
of  typhoid  patients,  the  agglutinins  may  often  be  found  in  dila- 
tions of  1  :  100  and  over.  It  is  interesting  to  note  that  irrespec- 
tive of  the  agglutinin  contents  of  any  given  serum,  there  may 
occasionally  be  noted  differences  in  the  agglutinability  of  various 
typhoid  cultures,  a  point  which  is  practically  important  in  the  choice 
of  a  typhoid  culture  for  routine  diagnosis  work.  Weeny  ^  has 
called  attention  to  the  fact  that  bacilli  which  do  not  readily  agglutinate 
when  directly  cultivated  from  the  body,  may  often  be  rendered  more 
sensitive  to  this  reaction  by  several  generations  of  cultivation  upon 
artificial  media.    Walker  has  noted  ^  a  loss  of  agglutinability  if  the  baciUi 


»  Weeny,  Brit.  Med.  Jour.,  1889. 

« Walker,  Jour,  of  Path,  and  Bact.,  1892;  Totsuka,  Zeit.  f.  Hyg.,  xlv,  1903. 


420 


PATHOGENIC   MICROORGANISMS 


are  cultivated  in  immune  serum.  A  similar  alteration  in  the  agglutin- 
abilit}^  of  typhoid  bacilli  was  noted  by  Eisenberg  and  Volk  *  when  they 
subjected  the  microorganism  to  moderate  heat  or  to  weak  acids  such  as 
f  HCl. 

The  practical  application  of  agglutination  to  bacteriological  work  is 
found,  as  in  the  case  of  the  bactericidal  substances,  in  the  identification 
of  suspected  typhoid  bacilli,  and  in  the  diagnosis  of  typhoid  fever. 

When  it  is  desired  to  determine  by  means  of  agglutination  whether 
or  not  a  given  bacillus  is  a  typhoid  bacillus,  mixtures  may  be  made 
of  young  broth  cultures,  gr  preferably  of  emulsions  of  young  agar  cul- 
tures in  salt  solution,  with  dilutions  of  immune  serum.  The  tests  are 
made  microscopically  in  the  hanging-drop  preparation  or,  preferably, 
macroscopically  in  small  test  tubes.  In  all  cases  it  is  desirable  first  to 
determine  the  agglutinating  power  of  the  serum  when  tested  against 
a  known  typhoid  culture.  (For  detailed  technique,  see  chapter  on 
Technique  of  Serum  Reactions,  p.  250.) 

In  scientific  investigations,  specific  agglutinations  in  high  dilutions 
of  immune  serum  constitute  very  strong  proof  of  the  species  of  the  micro- 
organism and  may  often  furnish  much  information  as  to  the  biological 
relationships  between  similar  species.  It  is  found  in  immunizing  ani- 
mals with  any  given  strain  of  typhoid  bacilli,  that  there  are  formed 
the  "chief"  or  ''major"  agglutinins  which  are  specific  and  active 
against  the  species  used  in  immunization,  and  the  "group "  or  "  minor  " 
agglutinins,  active  also  against  closely  related  microorganisms.  The 
following  extract  from  a  table  will  serve  to  illustrate  this  point  in  the 
case  of  typhoid  and  allied  bacilli. 


• 

Highly  Immune  Typhoid  Serum. 

1:100 

1:250 

1  :500 

1  : 1,000 

1  :  2,500 

B.  tvph 

+ 

+ 

+ 
+ 

+ 
+ 

+ 

+ 

B .  paratyph.  (Schottmiiller) 

B.  enteritidis ,    . 

B.  coli  communis. . . . 

The  sera  of  most  adult  normal  animals  and  human  beings  usually 
contain  a  small  amount  of  agglutinin  for  these  bacilli.  Immuniza- 
tion with  the  typhoid  bacillus,  while  increasing  chiefly  the  agglutinin 

^Eisenberg  und  Volk,  Zeit.  f.  Hyg.,  xlv,  1903. 


BACILLUS  OF  TYPHOID  FEVER  ^1 

for  this  bacillus  itself,  also  to  a  slighter  extent  increases  the  group  ag- 
glutinins for  other  closely  allied  species.  That  these  group  agglu- 
tinins are  separate  substances  and  not  merely  a  weaker  manifestation 
of  the  action  of  the  typhoid  agglutinin  itself  upon  these  other  micro- 
organisms, may  be  demonstrated  by  the  experiments  of  agglutiniiL 
absorption.     (See  section  on  Agglutinins,  page  234.) 

In  the  clinical  diagnosis  of  typhoid  fever,  the  phenomenon  of 
agglutination  was  first  utilized  by  Widal.^  This  observer  called  at- 
tention to  the  fact  that  during  the  last  part  of  the  first  or  the  earlier 
days  of  the  second  week  of  typhoid  fever,  as  well  as  later  in  the  dis- 
ease and  in  convalescence,  the  blood  serum  of  patients  would  cause 
agglutination  of  typhoid  bacilli  in  dilutions  of  1 :  10,  or  over,  whereas 
the  serum  of  normal  individuals  usually  exerted  no  such  influence. 
Upon  this  basis  he  recommended,  for  the  diagnosis  of  the  disease,  the 
employment  of  a  microscopic  agglutiv"iation  test  carried  out  by  the 
usual  hanging-drop  technique.  The  reaction  of  Widal  is,  at  present, 
widely  depended  upon  for  diagnostic  purposes  and  although  not  uni- 
versally successful,  owing  to  irregularities  in  agglutinin  formation 
in  some  patients  and  because  of  differences  in  agglutinability  of  the 
cultures  employed,  it  is  nevertheless  of  much  value.  The  fact  that 
the  recent  work  of  Hooker  and  of  Weiss  has  shown  that  typhoid  bacilli 
differ  in  antigenic  properties,  and  may  on  the  basis  of  agglutinatioii 
and  agglutinine  absorption  be  divided  into  a  number  of  groups,  will 
necessitate  the  use  of  several  cultures  for  Widal  reactions.  The  orig- 
inal conclusions  as  to  the  dilutions  of  the  serum  which  must  be  em- 
ployed, have,  however,  necessarily  been  modified.  Owing  to  the;  fact 
that  Gruber,^  Stern,^  Frank^l,^  and  a  number  of  others  have  found  that 
occasionally  normal  serum  will  give  rise  to  agglutination  of  typhmd 
bacilli  in  dilutions  exceeding  1 :  10,  it  has  been  found  necessary,  when- 
ever making' a  diagnostic  test,  to  make  several  dilutions,  the  ones  most 
commonly  employed  being  1:20,  1:40,  1:60,  and  1:80.  The  wide 
application  of  the  method  has  given  rise  to  the  development  of  a^ 
number  of  technical  procedures,  all  of  them  devised  with  a  view 
toward  simplification.  In  ordinary  hospital  work,  it  is  most  con- 
venient to  keep  on  hand  upon  slant  agar,  a  stock  typhoid  culture,  the 
agglutinability  of  which  is  well  known.  From  this  stock  culture,  fresh 

^  Widal,  Bull,  de  la  soc.  med.  des  hopit.,  vi,  1896;  Widal  et  Sicard,  Ann.  de 
I'inst.  Pasteur,  xi,  1897. 

2  Gruber,  Verhand.  Congr.  f.  inn.  Med,,  Wiesbaden,  1896. 

*Stem,  Gent.  f.  inn.  Med.,  xlix,  1896.    |,    » Frankel,  Deut.  med.  Woch.,  ii,  1897o 


422  PATHOGENIC  MIRCOORGANISMS 

inoculations  upon  neutral  bouillon  should  be  made  each  day,  so  that  a 
young  broth  culture  may  always  be  on  hand  to  furnish  actively  motile, 
evenly  distributed  bacteria.  These  bouillon  cultures  may  be  grown 
for  from  six  to  eight  hours  at  incubator  temperature  or  for  from  twelve 
to  eighteen  hours  at  room  temperature.  The  temperatures  at  which 
the  broth  cultures  are  kept  must  depend,  to  a  certain  extent,  upon  the 
peculiarities  of  the  typhoid  bacillus  employed,  since  some  strains  are 
rather  more  actively  motile  and  furnish  a  more  suitable  emulsion  if  kept 
at  a  temperature  lower  than  37.5°  C.  A  false  clumping  in  the  broth 
cultures  due  to  a  too  high  acidity  of  the  bouillon  or  a  too  prolonged 
incubation,  must  be  carefully  guarded  against.  It  is  also  possible  to 
use  for  this  test  an  emulsion  of  typhoid  bacilli  prepared  by  rubbing  up  a 
small  quantity  of  a  young  agar  culture  in  salt  solution.  Uniformity 
in  the  preparation  of  broth  cultures  or  of  emulsions  should  be  observed, 
since  the  quantitative  relationship  between  typhoid  bacilli  and  agglu- 
tinins will  markedly  affect  the  completeness  or  incompleteness  of  the 
reaction.  In  high  dilutions  an  excess  of  typhoid  bacilli  may  bring  about 
complete  absorption  of  all  the  agglutinins  present,  without  agglutinat- 
ing all  the  microorganisms. 

The  blood  of  the  patient  to  be  used  for  a  Widal  test  may  be  obtained 
in  a  number  of  ways.  The  most  convenient  method  is  to  bleed  the  pa- 
tient from  the  ear  or  finger  into  a  small  glass  capsule,  in  the  form  of  that 
used  in  obtaining  blood  for  the  opsonin  test,  or  into  a  small  centrifuge 
tube.  About  0.5  to  1  c.c.  is  amply  sufficient.  These  capsules  or  tubes, 
after  clotting  of  the  blood,  may  be  placed  in  the  centrifuge  which  in  a 
few  revolutions  will  separate  clear  serum  from  clot. .  The  dilutions  of 
the  serum  are  then  made.  It  is  best  to  use  sterile  physiological  salt  solu- 
tion as  a  diluent,  but  neutral  broth  may  be  used.  The  dilutions  may  be 
made  either  by  means  of  an  ordinary  blood-counting  pipette  or  by  means 
of  a  capillary  pipette  upon  which  a  mark  with  a  grease  pencil,  made 
about  an  inch  from  the  tip,  furnishes  a  unit  of  measure,  and  upon 
which  suction  is  made  by  means  of  a  rubber  nipple.  It  is  convenient 
to  have  at  hand  a  small  porcelain  palette  such  as  that  used  by  painters, 
in  which  the  various  cup-like  impressions  may  be  utilized  to  contain  the 
various  dilutions.  Dilutions  of  the  serum  are  made,  ranging  from  1  :  10 
to  1  :  50.  A  drop  of  each  of  these  dilutions  is  mixed  with  a  drop  of  the 
typhoid  culture  or  emulsion  upon  the  center  of  a  cover-slip  and  the  cover- 
slip  inverted  over  a  hollow  slide.  A  control  with  normal  serum  and 
the  same  culture  should  always  be  made  and  also  one  with  the  culture 
alone  to  exclude  the  possibility  of  spoHtaneous  clumping.     Mixture 


BACILLUS  OF  TYPHOID  FEVER  423 

with  the  typhoid  culture,  of  course,  each  time  doubles  the  dilutions 
so  that,  for  instance,  a  drop  of  serum  dilution  1  :  10,  plus  a  drop  of 
the  typhoid  culture,  gives  the  final  dilution  of  1  :  20.  The  prepara- 
tions may  be  examined  with  a  high  power  dry  lens  or  an  oil  im- 
mersion lens.  In  a  positive  reaction,  the  baciUi,  which  at  first  swim 
about  actively,  singly  or  in  short  chains,  soon  begin  to  gather  in  small 
groups  and  lose  much  of  their  activity.  Within  one-half  to  one  hour, 
they  will  be  gathered  in  dense  clumps  between  which  the  fluid  is  clear 
and  free  from  bacteria,  and  only  upon  the  edges  of  the  agglutinated 
masses  may  slight  motility  be  observed.  The  degree  of  dilution  and 
the  time  of  exposure  at  which  such  a  reaction  may  be  regarded  as  of 
specific  diagnostic  value,  have  been  largely  a  matter  of  empirical  de- 
termination. It  is  generally  accepted  at  present  that  complete  agglu- 
tination within  one  hour  in  dilutions  from  1  :  40  to  1  :  60  is  definite 
prcof  of  the  existence  of  typhoid  infection.  Exceptions,  however,  to 
this  rule  may  occur.  Agglutinations  of  typhoid  bacilli  in  dilutions  of 
1  :  40,  and  over,  have  occasionally  been  observed  in  cases  of  jaundice 
and  of  tuberculosis,  and  these  conditions  must  occasionally  be  consid- 
ered, though  their  importance  was  formerly  exaggerated. 

The  method  of  making  the  Widal  test  from  a  drop  of  whole  blood, 
dried  upon  a  slide,  is  not  to  be  recommended,  as  accuracy  in  dilution 
by  this  method  is  practically  impossible. 

As  stated  above,  the  agglutinin  reaction  rarely  appears  in  typhoid 
fever  before  the  beginning  of  the  second  week.  It  may  continue  during 
convalescence  for  as  long  as  six  to  eight  weeks  and  occasionally,  in  cases 
where  there  is  a  chronic  infection  of  the  gall-bladder,  a  Widal  reac- 
tion may  be  present  for  years  after  an  attack. 

For  very  exact  work,  even  in  clinical  cases,  the  microscopic  agglu- 
tination method  may  be  replaced  by  macroscopic  agglutination,  ac- 
cording to  the  technique  described  in  another  section  (page  229). 

In  order  to  avoid  both  the  necessity  of  keeping  alive  typhoid  cultures 
for  routine  agglutination  tests  and  also  to  preclude  the  danger  of  in- 
fection by  the  use  of  living  culture,  Ficker  ^  has  recommended  the  use 
of  typhoid  bacilli  killed  by  formalin.  This  method  has  no  advan- 
tages for  practical  purposes  and  in  scientific  bacteriological  work  it  is, 
of  course,  not  to  be  considered  in  comparison  with  the  other  exact 
methods. 

Precipitins, — The  investigations  of  Kraus  ^  in  1897,  by  which  the 

1  Ficker,  Bed.  klin.  Woch.,  xlviii,  1903.     « Kraus,  Wien.  klin.  Woch.,  xxxii,  1897. 


424  PATHOGENIC  MICROORGANISMS 

precipitins  were  discovered,  revealed  specific  precipitating  substances, 
among  others,  also  in  typhoid  immune  sera.  Since  Kraus'  original  in- 
vestigation, these  substances  have  been  studied  by  Norris  ^  and  others.^ 

Opsonins. — A  number  of  observers  have  shown  that  opsonins  specific 
for  the  typhoid  bacillus  are  formed  in  animals  immunized  with  these 
organisms.  Opsonins  are  formed  also  in  patients  suffering  from  typhoid 
fever,  but  exact  opsonic  estimations  in  all  these  cases  are  extremely 
difficult  because  of  the  rapid  lysis  which  these  bacteria  may  undergo 
both  in  the  serum,  and  intracellularly  after  ingestion  by  the  leucocytes. 
Klein  ^  has  attempted  in  part  to  overcome  this  difficulty  by  working 
with  dilutions  of  serum  and  at  the  same  time  using  comparatively  thick 
bacterial  emulsions  and  exposures  to  the  phagocytic  action  not  exceed- 
ing ten  minutes.  Chantemesse  *  has  claimed  that  the  opsonic  index  of 
typhoid  patients  was  increased  after  treatment  with  a  serum  obtained 
by  him  from  immunized  horses,  and  Harrison  ^  has  reported  similar 
results  in  patients  treated  by  a  modification  of  Wright's  method  of 
active  immunization.  Klein  claims  to  have  demonstrated  that  in 
typhoid-immune  rabbits,  after  five  injections,  the  opsonic  contents  of 
the  blood  were  increased  to  an  equal  extent  with  the  bactericidal  sub- 
stances. He  concludes  from  this  interesting  observation  that  it  may 
well  be  that  the  opsonins  are  quite  as  important  in  typhoid  immunity 
as  are  the  latter  substances. 

For  diagnostic  purposes  in  typhoid  fever  the  estimation  of  the  opsonic 
index,  so  far,  has  not  been  proven  to  be  of  great  value. 

Specific  Therapy  in  Typhoid  Fever. — The  failure  to  produce  a  soluble 
toxin  from  typhoid  cultures  has  naturally  so  far  precluded  the  possibility 
of  an  antitoxic  therapy,  such  as  that  which  has  been  successful  in  diph- 
theria. In  the  light  of  our  present  knowledge  of  the  poisonous  products 
of  the  typhoid  bacillus  it  seems  but  natural  that  attempts  by  earlier 
investigators  to  apply  the  principles  of  Behring's  work  to  typhoid  fever 
were  doomed  to  fail.  Attempts  to  employ  specific  bactericidal  and  bac- 
teriolytic sera  for  therapeutic  purposes  in  this  disease  have  also  been 
without  favorable  result. 

Active  Prophylactic  Immunization. — We  have  seen  that  work  by 
Pfeiffer  and  KoUe  and  later  by  many  others  has  shown  that  it  is  com- 

1  Nfftris,  Jour,  of  Inf.  Dis.,  I,  3,  1904. 

3  Barker  and  Cole,  22d  Ann.  Session,  Assn.  of  Amer.  Phys.,  Wash.,  1897* 

3  Klein,  Bull.  Johns  Hopkins  Hosp.,  1907. 

*  Chantemesse,  14th  Internatl.  Cong,  for  Hyg.,  Berlin,  1907.. 

^Karrispn,  Jour.  Royal  Army  Med.  Corps,  8,  1QQ7.. 


BACILLUS  OF  TYPHOID  FEVER  425 

paratively  easy  to  immunize  animals  actively  against  typhoid  infection 
by  the  systematic  injection  of  graded  doses,  at  first  of  dead  bacilli,  later 
of  fully  virulent  live  cultures.  Attempts  to.  apply  these  principles  pro- 
phylactically  have  been  made  recently  on  a  large  scale  by  Wright  and 
his  associates  upon  English  soldiers  in  South  Africa,  and  by  German 
observers  in  German  East  Africa. 

The  first  recorded  experiment  of  this  sort  which  was  done  upon  human 
beings  was  that  of  Pfeiffer  and  KoUe,*  who  in  1896  treated  two  in- 
dividuals with  subcutaneous  injections  of  an  agar  culture  of  typhoid 
bacilli  which  had  been  sterilized  at  56°  C.  The  first  injection  was  made 
with  two  milligrams  of  this  culture.  Three  or  four  hours  after  the  in- 
jection the  patient  suffered  from  a  chill,  his  temperature  gradually  rose 
to  105°  F.,  and  there  was  great  prostration  and  headache,  but  within 
twenty-four  hours  the  temperature  had  returned  to  normal. 

This  experiment  showed  that  such  injections  could  be  practiced  upon 
human  beings  without  great  danger. 

Simultaneously  with  the  work  of  Pfeiffer  and  Kolle,  Wright  ^  con- 
ducted similar  experiments  on  officers  and  privates  in  the  English  army. 

The  actual  number  of  persons  treated  directly  or  indirectly  under 
Wright's  ^  supervision  in  an  investigation  covering  a  period  of  over  four 
years  comprised  almost  one  hundred  thousand  cases.  The  methods 
employed  by  Wright  have  been  modified  several  times  in  minor  details; 
the  principles,  however,  have  remained  consistently  the  same.  In  the 
first  experiments  Wright  employed  an  agar  culture  three  weeks  old, 
grown  at  37°  C,  then  sterilized  at  a  temperature  below  60°  C,  and  pro- 
tected from  contamination  by  the  addition  of  five-tenths  per  cent  of 
carboUc  acid*.  Later,  Wright^  employed  bacilli  grown  in  a  neutral 
one-per-cent  pepton  bouillon  in  shallow  layers  or  flasks. 

Great  importance  is  attached  both  to  the  virulence  of  the  typhoid 
strain,  which  may  to  a  moderate  extent  be  standardized  by  passage 
through  guinea-pigs,  and  to  care  in  using  bw  temperatures  for  final 
sterilization.  The  temperature  recommended,  by  Harrison,^  is  52°  €).,, 
after  which  the  cultures  are  aarboUzed.. 

1  Pfeiffer.  nnd  KoUCf  Deut.  med.  Wocb.,  xxii,  1896;  xxiv,  1898. 

2  Wright,  Lancet,  Sept.,  1896. 

3  Wright  and  Semple,  Brit.  Med.  Jour.,  1897;  Wright  and  Leishman,  Brit.  Med. 
Jour;,  Jan.,  1900. 

*  Wright,  Brit.  Med.  Jou^r,,  1901;  Lancet,  Sept.,  1902;  Brit.  Medr  Jpuj*.,  Oct.,, 
1903. 

5  Uarrison,  Jour.  Iio3^^1 ,  Arpay ,  Medical  Corps^  1907, , 


426  PATHOGENIC  MICROORGANISMS 

It  is  nevertheless  extremely  difficult  to  tabulate  satisfactory  statis- 
tics from  a  mass  of  experiments  observed  by  a  large  number  of  indi- 
viduals. On  the  whole,  however,  it  seems  fair  to  state  that  advan- 
tageous results  followed  the  active  immunization  practiced  by  Wright. 
Wright's  own  estimation,  in  a  careful  attempt  to  present  the  subject 
fairly,  gives  a  reduction  of  the  morbidity  from  typhoid  fever  in  the 
British  army  of  fifty  per  cent,  and  a  reduction  of  the  mortality  of  those 
who  became  infected  in  spite  of  inoculations  of  fifty  per  cent  also. 
It  is  not  at  all  impossible  that  a  number  of  different  strains  will  have 
to  be  used  eventually  for  the  ideal  vaccine,  inasmuch  as  the  antigenic 
differences  which  have  been  recently  discovered  and  alluded  to  would 
make  it  seem  that  no  si»gle  strain  can  be  expected  to  produce  anti- 
bodies which  would  protect  against  all  other  strains.  It  is  not  impos- 
sible that  some  individual  strain  may  combine  the  antigenic  properties 
of  the  entire  group.    This,  however,  has  still  to  be  worked  out. 

The  method  of  Peiffer  and  Kolle  consists  in  the  injection  of  salt- 
solution  emulsions  of  fresh  agar  cultures  sterilized  at  60°  C.  The 
results  reported  were  in  general  favorable. 

Recent  extensive  tests  in  the  United  States  Army,  observed  by  Rus- 
sell,^ seem  to  have  removed  any  doubt  which  may  have  existed  as  to 
the  efficacy  of  prophylactic  typhoid  vaccination.  Russell's  statistics 
show  a  steady  decline  of  typhoid  in  the  U.  S.  Army  beginning  with  the 
introduction  of  compulsory  vaccination  in  1910.  In  1913  there  was 
but  one  case  among  over  80,000  men. 

The  method  at  present  employed  is  as  follows:  The  ''Rawlings" 
strain  of  typhoid,  obtained  from  Wright,  is  used.  "  Eightoen-hour  agar 
cultures  in  Kolle  flasks  are  washed  off  with  sterile  saline  to  an  approxi- 
mate concentration  of  one  billion  to  the  c.c.  The  suspension  is  killed 
at  53°  C.  for  one  hour  and  0.25  per  cent  tricresol  is  added.  Aerobic 
and  anaerobic  culture  controls  are  made  and  a  rabbit  and  mouse  inocu- 
lated to  insure  sterility.  For  immunization  3  to  4  doses  are  given 
ranging  in  quantity  from  500  million  to  one  billion  at  7  to  10  day 
intervals.  The  protection  probably  lasts  about  2  years,  though  this  is 
not  certain. 

Another  point  of  importance  in  this  connection  has  recently  been 
raised  by  Metchnikoff  and  Besredka.^  They  vaccinated  chimpanzees 
with  typhoid  bacilli  and  found  that  when  emulsions  of  the  clear  bac- 

1  Russell,  Am.  Jour,  of  Med.  Sc,  cxlvi,  1913.. 

2  Metchnikoff  and  Besredka,  Am.  de  Tinst.  Past.,  1911. 


BACILLUS  0^  TYPHOID  FEVE^  427 

teria  were  used,  protection  was  only  slight.  Better  results  were  ob- 
tained— that  is,  apparently  complete  protection  within  8  to  10  days — 
when  living  sensitized  bacteria  were  injected.  (Bacteria  which  had 
been  exposed  to  the  action  of  inactivated  immune  serum.)  Brough- 
ton  ^  has  applied  this  method  to  human  beings.  Gay  ^  has  also  pre- 
pared a  sensitized  dead  typhoid  vaccine  which  he  has  already  used  in 
a  considerable  number  of  cases.  It  will  take  some  time,  however,  before 
a  statistical  estimation  of  the  superiority  of  this  method  over  the  older 
vaccination  with  dead  bacteria  will  be  possible. 

Attempts  have  recently  been  made  to  treat  active  typhoid  fever  by 
intravenous  injections  of  sensitized  vaccines.  The  cases  are  as  yet  too 
few  to  permit  final  judgment. 

BACILLUS  FECALIS  ALKALIGENES 

In  1896  Petruschky  ^  described  a  bacillus  which  is  a  not  infrequent 
inhabitant  of  the  human  intestine,  being  found  chiefly  in  the  lower 
part  of  the  small  intestine  and  the  large  intestine.  This  organism, 
which  he  called  Bacillus  fecalis  alkaligenes,  is  of  little  pathogenic  im- 
portance, although  Neufeld  states  that  he  has  seen  a  case  of  severe 
gastroenteritis  in  which  the  watery  defecations  contained  this  bacillus 
in  almost  pure  culture.  As  a  rule,  however,  this  organism  cannot  be 
regarded  as  pathogenic,  and  is  important  chiefly  because  of  the  ease 
with  which  it  may  be  mistaken  for  Bacillus  typhosus. 

Bacillus  fecalis  alkaligenes  is  an  actively  motile,  Gram-negative 
bacillus, '  possessing,  like  the  typhoid  bacillus,  numerous  peritrichal 
flagella.  On  the  ordinary  culture  media  it  grows  like  the  typhoid 
bacillus.  It  does  not  coagulate  milk.  It  produces  no  indol,  and  on 
sugar  media  in  fermentation  tubes  produces  no  acid  or  gas.  On 
potato,  its  growth,  while  somewhat  heavier  than  that  of  the  typhoid 
bacillus,  is  not  sufficiently  so  to  permit  easy  differentiation.  It  differs 
from  Bacillus  typhosus  in  that  it  produces  no  acid  on  any  of  the  sugar 
media,  and  is  therefore  easily  differentiated  by  cultivation  upon  Hiss 
serum-water  media  or  on  pepton  waters  containing  sugars.  On  the 
Hiss  semi-solid  tube-medium  Bacillus  fecalis  alkaligenes,  while  cloud- 
ing the  medium  throughout,  grows  most  heavily  on  the  surface,  where, 
eventually,  it  forms  a  pellicle. 

1  Broughton,  C.  R.  de  I'Acad.  des  Sc,  cliv,  1911. 

2  Gay,  Arch,  of  Int.  Med.,  1914. 

»  ?eiruschky.  Cent.  f.  Bakt.,  I,  xix,  1896- 


CHAPTER  XXVIII 

"BACILLI  OF  THE  COLON-TYPHOID-DYSENTERY  GROUP 

,  ifiordinued) 

BACILLI  INTERMEDIATE  BETWEEN  THE  TYPHOID  AND  COLON 

ORGANISMS 

{Bacilli  of  Meat  Poisoning  and  Paratyphoid  Fever) 

There  is  an  extensive  group  of  Gram-negative  bacilli  which  be- 
cause of  their  morphology,  cultural  behavior,  and  pathogenic  properties, 
are  classified  as  intermediate  between  the  colon  and  the  typhoid  types. 
The  microorganisms  belonging  to  this  group  have  been  described,  most 
of  them,  within  the  last  fifteen  years,  but  few  of  them  have  been  fully 
identified  with  one  another.  They  have  been  variously  designated  as  thp 
"hog-cholera  group,"  "the  enteritidis  group,"  the  "paracolon  group" 
or  "paratyphoid  group,"  because  of  the  pathological  conditions  with 
which  the  chief  members  under  investigation  have  been  found  associated. 

Attempts  to  systematize  the  group  by  the  comparative  study  of  a 
large  number  of  its  members  have  been  made,  notably  by  Buxton^ 
and  by  Durham,^  and  the  work  of  these  writers,  based  on  cultural  and 
agglutinative  studies,  has  added  materially  to  our  knowledge  of  these 
organisms. 

The  microorganisms  of  this  group  are  morphologically  indistinguish- 
able from  the  colon  and  typhoid  bacilli.  They  are  Gram-negative  and 
possess  flagella.  Their  motility  is  variable,  but  usually  approaches 
that  of  the  typhoid  bacilli  in  activity.  They  correspond,  furthermore, 
to  the  two  other  groups  in  their  cultural  characteristics  upon  broth,  agar, 
and  gelatin.  On  'potato,  they  vary,  some  of  them  approaching  in  deli- 
cacy the  typhoid  growth  upon  this  medium,  others  more  closely 
approximating  the  heavy  brownish  growth  of  B.  coH.  Indol  is  rarely 
formed  by  them,  though  this  has  not  been  absolutely  constant  in  all 
descriptions.    As  a  group,  they  are  easily  distinguished  from  Bacillus 

1  Buxton,  Jour.  Med,  Res.,  N.  S.,  iii,  1900.     » Durham,  Jour.  Exper.  Med.,  v,  1901. 

428 


BACILLI  BETWEEN  TYPHOID  AND  COLON  ORGANISMS       429 

typhosus  on  the  one  hand,  and  from  Bacillus  coli  on  the  other,  by  the 
following  simple  reactions  tabulated  by  Buxton.^ 


B.  coli. 

Intermediates. 

B.  typhosus. 

Coagulation  of  milk 

+ 
+ 
+ 
+ 

+ 

_ 

Production  of  indol 

Fermentation  of  lactose  with  gas 

Fermentation  of  dextrose  with  gas 

Agglutination  in  typhoid-immune  serum. 

+ 

Characteristics  of  the  three  groups  as»shown  by  fermentation  tests 
follow: 


Gas  upon 
Dextrose. 

Gas  upon 
Lactose. 

Gas  upon 
Saccharose. 

Gas  on 
Dulcit. 

B  typhosus     .     .      

+ 
+ 
+ 
+ 
+ 

+ 

+ 

+ 

•          + 

+ 
+ 

Intermediates 

B.  coli  communis 

+ 

B.  coli  communior 

B.  acidi  lactici 

B.  lactis  aerogenes  ^ 

Pathogenically,  the  bacilli  of  this  "intermediate  group*'  have  attracted 
attention  chiefly  in  connection  with  meat  poisoning,  and  with  protracted 
fevers  indistinguishable  from  mild  typhoidal  infections. 

In  1888,  Gartner  ^  described  a  bacillus  which  he  isolated  from  the 
meat  of  a  cow,  the  ingestion  of  which  had  produced  the  symptoms  of 
acute  gastrointestinal  catarrh  in  57  people.  One  of  these  died  of  the 
disease  and  the  bacilli  could  be  demonstrated  in  the  spleen  and  blood  of 
the  patient. 

This  bacillus,  called  Bacillus  enteritidis  by  Gartner,  was  actively 
motile,  formed  no  indol,  but  produced  gas  in  dextrose  media.  Acute 
gastrointestinal  symptoms  could  be  induced  by  feeding  the  organisms 
to  mice,  guinea-pigs,  rabbits,  and  sheep,  and  the  bacilli  could  be  recov- 
ered from  the  infected  animals.  An  interesting  observation,  which 
has  since  become  important  in  characterizing  the  group  of  these  bacilli 
concerned  in  meat  poisoning,  was  the  fact  that  the  bacterial  bodies 
themselves  were  foimd  by  Gartner  to  be  extremely  toxic,  containing  a 
poison  which,  in  contradistinction  to  the  endotoxins  of  many  other 
microorganisms,  was  extremely  resistant  to  heat.  Sterilized  cultures 
showed  the  same  pathogenic  effects  as  the  living  bacilli.     Epidemics 

1  Buxton,  loc.  cit.  2  Jackson. 

*  Gartner f  Corresp.  Bl.  d.  Aerzt.  Vereins,  Turingen,  1888.  ' 


430  PATHOGENIC  MICROORGANISMS 

of  meat  poisoning  similar  to  the  one  described  by  Gartner,  in  which 
similar  bacteria  were  isolated,  were  those  described  by  Van  Ermen- 
gem,^  occurring  at  Morseele  in  1891,  the  one  described  by  Holst,^  the 
Rotterdam  epidemic  described  by  Poels  and  Dhont,^  the  one  described 
by  Basenau,  and  many  others. 

Bacillus  Morseele  of  Van  Ermengem,  Bacillus  hovis  morhificans  of 
Basenau,*  and  bacilli  isolated  in  similar  epidemics  by  others,  are,  ex- 
cept for  slight  differences  in  minor  characteristics,  almost  identical 
with  Gartner's  microorganism. 

In  1893,  Theobald  Smith  and  Moore  ^  noted  a  great  similarity  be- 
tween the  so-called  hog-cholera  bacillus,  the  bacilli  of  the  Gartner 
group,  and  Bacillus  typhi  murium  isolated  by  Loeffler.  These  ob- 
servers first  used  the  term  *' hog-cholera"  group  for  the  organisms 
under  discussion. 

In  1899  Reed  and  Carroll®  noted  that  Bacillus  icteroides,  asso- 
ciated by  Sanarelli  with  yellow  fever,  was  culturally  similar  to  the 
bacillus  of  hog  cholera. 

Meanwhile,  other  observers  had  been  isolating  bacilli,  similar  to 
those  spoken  of  above,  from  cases  of  protracted  fevers  in  human 
beings,  often  closely  simulating  typhoid  infections.  The  first  cases  of 
this  kind  on  record  were  those  of  Achard  and  Bensaude.'^ 

In  1897,  Widal  and  Nobecourt  ^  described  a  bacillus  which  they  had 
isolated  from  an  esophageal  abscess  following  typhoid  fever,  which 
closely  resembled  Bacillus  psittacosis  of  Nocard,®  and  which,  following 
a  nomenclature  previously  suggested  by  Gilbert,-^^  they  designated  the 
paracolon  bacillus.  This  microorganism,  isolated  from  a  parrot  by 
Nocard,  showed  a  close  resemblance  to  bacilli  of  the  Gartner  group. 

There  are  a  large  number  of  apparently  nonpathogenic  organisms 
sometimes  referred  to  as  paratyphoid  C,  but  better  perhaps  as  ''hetero- 
genous types,''  which  are  culturally  identical  with  the  paratyphoid 

*  Van  Ermengem,  Bull.  Acad.  d.  m^d.  de  Belgique,  1892;  "Trav.  de  lab.  de 
Puniv.  de  Gand,"  1892. 

2  Hoist,  Ref .  Cent.  f.  Bakt.,  xvii,  1895. 

'  Poels  und  Dhont,  Holland  Zeit.  f.  Tierheilkunde,  xxiii,  1894. 

*  Basenau,  Arch,  f .  Hyg.,  xx,  1894. 

"  Th.  Smith  and  Moore,  U.  S.  Bureau  of  Animal  Industry  Bull.,  vi,  1894. 
«  Reed  and  Carroll,  Medical  News,  Ixxiv,  1899. 

'  Achard  and  Bensaude,  Bull,  de  la  soc.  d.  h6pitaux  de  Paris,  Nov.,  1906. 
8  Widal  et  Nobecourt,  Semaine  m€d.,  Aug.,  1897. 
^  Nocard,  Ref.  Baumgarten's  Jahresb.,  1896. 
"  Gilbert,  Semame  m6d.,  1895. 


BACILLI  BETWEEN  TYPHOID  AND  COLON  ORGANISMS      431 

but  do  not  agglutinate  in  either  paratyphoid  A  or  B  sera.  An  anti- 
serum produced  with  these  types  usually  reacts  with  the  homologous 
strain  only.  Such  organisms  have  been  studied  by  Krumwiede  and 
many  others,  including  ourselves.  Strains  recently  isolated  at  this 
laboratory  came  from  cases  of  nephritis,  German  measles,  jaundice, 
and  the  stools  of  healthy  soldiers,  done  as  a  matter  of  routine. 

In  1898,  Gwyn  ^  reported  a  case  at  the  Johns  Hopkins  Hospital, 
which  presented  all  the  symptoms  of  typhoid  fever,  but  lacked  serum 
agglutinating  power  for  Bacillus  typhosus.  From  the  blood  of  the  pa- 
tient, Gwyn  isolated  an  organism,  with  cultural  characteristics  similar 
to  those  of  the  Gartner  bacillus,  which  he  called  a  ' '  paracolon  bacillus. ' ' 
This  bacillus  was  agglutinated  specifically  by  the  serum  of  the  patient. 

Cushing,^  in  1900,  isolated  a  similar  microorganism  from  a  costo- 
chondral  abscess,  appearing  during  convalescence  from  typhoid  fever. 

In  the  same  year,  Schottmiiller  ^  reported  five  cases  from  which 
similar  bacilli  were  isolated.  Careful  cultural  studies  of  the  microor- 
ganisms here  obtained  showed  that  they  could  be  divided  into  two  simi- 
lar, yet  distinctly  different  types,  one  of  them,  the  ''Miiller*'  organism, 
approaching  closely  to  the  typhoid  type,  especially  in  its  growth  upon 
potato;  the  other,  the  '*Seeman''  type,  .corresponding  more  closely  to 
the  Gartner  bacilli.  Similar  cases  were  reported  by  Kurth,*  Buxton 
and  Coleman,^  Libman,^  and  others. 

The  two  types  of  organisms,  paratyphoid  A  and  B,  described  by 
Schottmiiller  and  studied  by  many  other  observers,  can  be  culturally 
differentiated,  though  not  without  difficulty. 

Type  A  is  more  delicate  in  its  growth  on  various  media  than  B, 
growing  with  almost  invisible  growth  on  potato,  and  differing  from 
typhoid  in  its  gas  formation  on  dextrose  broth.  Milk  is  not  coagu- 
lated, but  remains  turbid,  not  being  finally  cleared  by  solution  of  the 
casein  as  in  similar  cultures  of  type  B.  Lactose  whey  is  acidified  and 
remains  acid.  This  organism  has  been  isolated  from  the  normal  intes- 
tines of  animals  by  Morgan.'^  Kutscher  for  this  reason  suggests  that 
essentially  and  except  in  rare  instances  this  organism  is  a  non-patho- 

*  Gwyn,  Johns  Hopkins  Hosp.  Bull.,  1898. 

2  Gushing,  Johns  Hopkins  Hosp.  Bull.,  1900. 

^  Scholtmuller,  Deut.  med.  Woch.,  1900;  Zeit.  f.  Hyg.,  xxvi. 

*  Kurth,  Deut.  med.  Woch.,  1901. 

6  Buxton  and  Goleman,  Proc.  N.  Y.  Pathol.  Soc,  Feb.,  1902. 

« lAbman,  Jour.  Med.  Res.,  N.  S.,  iii,  1902. 

'  Morgan,  cited  from  Kutscher,  KolU  und  WassermanUf  Handbuch.  Erganzungs,  I. 


432  PATHOGENIC  MICROORGANISMS 

genie  saprophyte.  Recently  Krumwiede,  Pratt,  and  Kohm  have  shown 
that  the  members  of  this  group  and  some  of  the  so-called  paratyphoid 
C  group  may  be  distinguished  from  all  the  other  paratyphoids  by 
inability  to  ferment  xylose,  and  Weiss  has  shown  paratyphoid  A  may 
be  subdivided  on  the  basis  of  antigenic  properties. 

Type  B  grows  more  heavily  on  all  media  than  A,  especially  on 
potato  (though  this  is  irregular).  Milk  is  slightly  acidified  at  first, 
but  eventually  is  rendered  alkaline  and  cleared,  possibly  by  casein 
solution. 

Subdivision  of  this  group  was  made  by  Weiss  and  others  on  the 
basis  of  inosite  fermentation.  Eventual  differentiation  must  be  made 
by  agglutination. 

The  diseases  caused  by  these  bacteria  may  be  divided  as  follows : 

I.  Those  which  fall  into  the  category  of  meat  poisoning  with  sudden 
onset  of  gastroenteric  symptoms  following  ingestion  of  meat;  and 

II.  Those  in  which  the  disease  simulates  a  mild  typhoid  fever,  dif- 
fering from  ttis  only  by  the  absence  of  the  specific  agglutination. 

The  differential  diagnosis  between  the  second  type  of  case  and  true 
typhoid  fever  may  be  extremely  difficult.  In  contradistinction  to 
true  typhoid  the  temperature  reaction  of  this  case  may  set  in  more 
abruptly  and  remain  more  irregular  throughout  the  disease.  Gastric 
symptoms,  vomiting,  and  nausea  are  often  more  prominent  than  in 
typhoid  fever  and  enlargement  of  the  spleen  is  less  regularly 
present.  Owing  to  the  low  mortality  of  paratyphoid  fever  (in 
120  cases  observed  by  Lentz  ^  less  than  4  per  cent,  and  in  many 
other  smaller  epidemics  no  deaths  have  occurred) ,  we  have  remained 
relatively  ignorant  concerning  the  pathologic  anatomy  of  the  dis- 
ease. Longcope  ^  observed  a  case,  fatal  after  two  weeks  of  illness, 
in  which  there  was  no  enlargement  of  Peyer's  patches  and  no  sign  of 
even  beginning  ulceration.  Most  other  observers  have  also  found  less 
involvement  of  the  lymphatics  of  the  bowel  than  is  found  in  typhoid 
fever.  During  the  disease  the  bacteria  can  often  be  cultivated  from 
the  blood,  and  the  serum  of  the  patient  may  agglutinate  specifically 
paratyphoid  strains.  In  this  way  the  diagnosis  can  often  be  made. 
Libmann  ^  has  isolated  the  organism  from  the  fluid  aspirated  from  the 
gall  bladder  in  a  case  operated  on  for  cholecystitis. 

Most  of  these  microorganisms  possess  pathogenicity  for  mice,  guinea- 

^  Lentz,  Klin.  Jahrb.,  xiv,  1914. 

2  Longcope,  Amer.  Jour,  of  Med.  Sciences,  cxxiv,  1902. 

*  Libmann,  Jour,  of  Med.  Res.,  viii,  1902. 


BACILLI  BETWEEN  TYPHOID  AND  COLON  ORGANISMS     433 

pigs,  and  rabbits,  which  exceeds  that  of  the  colon  or  typhoid  bacilli. 
A  number  of  the  bacilli  of  this  group,  furthermore,  especially  those  most 
closely  similar  to  the  original  B.  enteritidis  of  Gartner,  contain  an  endo- 
toxin which  shows  a  high  resistance  to  heat,  which  may  explain  the  fact 
that  illness  has  occasionally  followed  the  ingestion  of  infected  meat  even 
after  preparation  by  cooking. 

Bacteriological  correlation  of  these  bacilli  has  been  attempted,  as 
stated  above,  by  Durham  and  by  Buxton,  and  more  recently  by 
Kutscher  and  Meinicke.^  The  subject  is  a  difficult  one  and  for  ultimate 
clearness  will  require  much  further  work. 

Harding  and  Ostenberg  ^  have  examined  a  series  of  organisms  of  the 
intermediate  group  on  various  sugars,  and  find  that  by  the  use  of 
xylose  and  arabinose  three  definite  groups  can  be  established. 

I.  Those  making  aldehyd  (red)  on  fuch'oin-sulphite  agar  with  both 
arabinose  and  xylose — both  Schottmiiller  types  A  and  B  and  strains 
of  Bacillus  enteritidis. 

II.  Red  on  arabinose  and  not  on  xylose — typhi  murium,  para- 
typhoid Gwyn,  paratyphoid  Loomis,  and  three  others. 

III.  Red  on  xylose  and  not  on  arabinose — B.  hog  cholera. 

This  work  was  carefully  carried  out  and  may  possibly  point  toward 
an  ultimate  classification.  However,  the  strains  employed  were  too 
few  to  permit  definite  conclusions  at  present. 

Durham,^  on  the  basis  of  cultural  and  agglutinative  studies,  has 
formulated  a  classification  of  the  Gram-negative  bacilli  of  the  typhoid- 
colon  and  aUied  groups,  which,  though  hardly  final,  aids  considerably 
in  throwing  light  upon  the  interrelationships  of  the  various  species. 
Durham's  divisions  are  as  follows: 

Division  I.   Typhoid-like  Morphology  (motile). 

A.  No  sugars  fermented.    Type  B.  fecalis  alkaligenes. 

B.  Acid  in  dextrose,  but  no  gas.  Type  B.  typhosus.  Agglutination 
in  typhoid  serum. 

C.  Acid  in  dextrose,  but  gas  only  when  other  constituents  are  favor- 
able. No  acid  or  gas  from  lactose  or  saccharose.  No  agglutination  in 
typhoid  serum.  Includes  Bacillus  "Gwyn"  and  Bacillus  "0"  of 
Gushing. 

D.  Acid  and  gas  from  dextrose.     No  acid  or  gas  from  lactose  or. 

^  Kutscher  und  Meinicke,  Zeit.  f.  Hyg.,  lii,  1906. 
2  Harding  and  Ostenberg,  Jour,  of  Inf.  Dis.,  ii,  1912. 
'  Durham^  loc.  cit. 


434  PATHOGENIC    MICROORGANISMS 

saccharose.  Grows  more  rapidly  than  typhoid.  No  agglutination  in 
colon-immune  serum.  Slight  reaction  with  some  typhoid  sera.  Includes 
Gartner's  B.  enteritidis,  B.  Morseele,  Gunther's  meat-poisoning  bacillus, 
hog  cholera  bacillus,  B.  psittacosis,  B.  morbificans  bo  vis,  Durham's 
Bacillus  "A,"  B.  typhi  murium. 

Division  II.   Colon-like  Morphology  (motile). 

E.  Acid  and  gas  from  dextrose,  none  from  lactose  or  saccharose. 
Rate  of  growth  and  colony  appearance  more  Uke  colon  than  typhoid. 

F.  Acid  and  gas  from  dextrose,  and  no  gas  from  lactose.  Types 
isolated  by  Durham. 

G.  Acid  and  gas  from  dextrose;  acid,  no  gas,  from  lactose.  Differ 
from  F  in  serum  reactions. 

H.  B.  coli  communis.  Acid  and  gas  from  dextrose  and  lactose;  none 
from  saccharose. 

/.  B.  coli  communior.  Acid  and  gas  from  dextrose,  lactose,  and 
saccharose. 

Division  III.  Non-motile.  Polysaccharide  splitters  (starch).  Type 
B.  lactis  aerogenes.  Includes  baciUi  of  mucosus  capsulatus  group,  and 
Friedlander's  bacillus. 


CHAPTER  XXIX 

BACILLI  OF  THE  COLON-TYPHOID-DYSENTERY  GROUP 

{Continued) 

THE  DYSENTERY  BACILLI 

Although  acute  dysentery  has  been  an  extremely  prevalent  disease, 
occurring  almost  annually  in  epidemic  form  in  some  of  the  Eastern  coun- 
tries and  appearing  sporadically  all  over  the  world,  its  etiology  was 
obscure  until  1898  when  Shiga  '  described  a  bacillus  which  he  isolated 
from  the  stools  of  patients  suffering  from  this  disease  in  Japan,  and  es- 
tablished with  scientific  accuracy  its  etiological  significance.  Since  the 
discovery  of  Shiga's  bacillus  a  number  of  other  bacilli  have  been  de- 
scribed by  various  workers,  all  of  which,  while  showing  slight  biological 
differences  from  Shiga's  microorganism,  are  sufficiently  similar  to  it 
culturally  and  pathogenically  to  warrant  their  being  classified  together 
with  it  in  a  definite  group  under  the  heading  of  the  "  dysentery  bacilli." 

The  manner  in  which  Shiga  made  his  discovery  furnishes  an  in- 
structive example  of  the  successful  application  of  modern  bacteriological 
methods  to  etiological  investigation.  Many  workers  preceding  Shiga 
had  attempted  to  throw  light  upon  this  subject  by  isolations  of  bacilli 
from  dysenteric  stools,  and  by  extensive  animal  inoculation.  Shiga, 
following  a  suggestion  made  by  Kitasato,  approached  the  problem  by 
searching  for  a  microorganism  in  the  stools  of  dysentery  patients  which 
would  specifically  agglutinate  with  the  serum  of  these  patients.  His 
labors  were  crowned  with  success  in  that  he  found,  in  thirty-six  cases, 
one  and  the  same  microorganism  which  showed  uniform  serum  agglu- 
tinations. Further,  he  found  that  this  bacillus  was  not  present  in  the 
dejections  of  patients  suffering  from  other  diseases  nor  in  those  of  normal 
men,  and  that  when  tested  against  the  blood  serum  of  such  people  it 
was  not  agglutinated. 

Morphology. — Shiga's  bacillus  is  a  short  rod,  rounded  at  the  ends, 

» iS/iigia,  Cent.  f.  Bakt.,  xxiii,  1898;  ibid.,  xxiv,  1898;  Deut.  med.  Woch.,  xliii, 
xliv,  and  xlv,  1901. 

435 


436  PATHOGENIC  MICROORGANISMS 

morphologically  very  similar  to  the  typhoid  bacillus,  and,  like  it, 
inclined  to  involution  forms.  The  organism  generally  occurs 
singly,  more  seldom  in  pairs.  It  is  decolorized  by  Gram's 
method  of  staining.  With  the»ordinary  anilin  dyes  it  stains  easily, 
showing  a  tendency  to  stain  with  slightly  greater  intensity  at 
the  ends.  The  organism  is  an  aerobe  and  facultative  anaerobe. 
Although  described  at  first  by  Shiga  as  being  motile,  its  motility 
has  not  been  satisfactorily  proven,-  and  most  observers  agree  in 
denying  the  presence  of  flagella  and  affirming  the  complete  absence 
of  motility. 

Cultural  Chaxacteristics. — On  agar  the  colonies  are  not  characteristic, 
resembling  those  of  the  typhoid  bacillus. 

On  gelatin,  the  colonies  appear  very  much  like  typhoid  colonies  and 
the- gelatin  is  not  liquefied. 

On  'potato,  the  growth,  like  that  of  typhoid,  is  at  first  not  visible,  but 
after  about  a  week  turns  reddish  brown. 

In  broth,  there  is  clouding,  with  moderate  deposits  after  some  days. 
No  pellicle  is  formed. 

Milk  is  not  coagulated.  Litmus  milk  shows  a  slight  primary  acidity, 
later  again  becoming  alkaline  and  taking  on  a  progressively  deeper  blue 
color. 

Indol  is  not  formed  in  pepton  water  by  all  varieties. 

No  gas  is  formed  in  media  containing  dextrose,  lactose,  saccharose, 
or  other  carbohydrate. 

While  not  delicately  susceptible  to  reaction^  the  bacillus  prefers 
slightly  alkaline  media.  . 

Shiga  differentiated  his  organism  from  the  typhoid  bacillus  chiefly 
by  supposed  differences  in  colony  characters  and  by  the  agglutination 
reaction. 

Following  the  work  of  Shiga,  a  large  number  of  investigators  turned 
Cheir  attention  to  the  subject  of  dysentery,  with  the  result  that  many 
new  forms  were  discovered  and  at  first  a  considerable  amount  of  con- 
fusion prevailed. 

Flexner  *  in  1899  investigated  dysentery  in  the  Philippines,  and 
isolated  a  bacillus  which,  he  considered,  corresponded  to  Shiga's 
organism. 

Strong  and  Musgrave/'  in  1900  described  a  bacillus  isolated  from 


^Flexner,  Phila.  Med.  Jour.,  vi,  1900,  and  Bull.  Johns  Hopkins  Hosp.,  xi,  1900. 
•  Strong  and  Musgrave,  Report  Surg.  Gen.  of  Army,  Washington,  1900. 


THE  DYSENTERY  BACILLI  437 

dysentery  cases  in  the  Philippines  which  was  essentially  like  that  of 
Flexner. 

Nearly  simultaneously  with  the  papers  of  Flexner  and  of  Strong  and 
Musgrave,  Kruse  ^  published  investigations  of  an  epidemic  of  dysentery 
occurring  in  Germany.  His  observations  were  of  the  greatest  importance 
and  largely  formed  the  starting  point  of  the  further  advances  which 
have  been  made  in  the  etiology  ^f  dysentery. 

Kruse's  organism  was  described  as  forming  colonies  on  gelatin  and 
agar,  practically  like  those  of  Bacillus  typhosus.  Like  this  bacillus,  no 
gas  was  formed  from  grape  sugar,  and  the  growth  in  milk  and  on  potato, 
and  even  in  Piorkowski's  urine  gelatin^  resembled  that  of  Bacillus 
typhosus.  According  to  Kruse,  this  organism  was  absolutely  with- 
out motility. 

In  1901  Kruse  ^  contributed  a  second  paper.  In  this,  besides  con- 
firming his  previous  observations,  he  described  another  class  of  organ- 
ism coming  from  cases  which  he  designated  as  "pseudo-dysentery  of 
insane  asylums."  In  the  case  of  one  patient,  and  at  two  autopsies,  he 
isolated  organisms  which  he  could  not  distinguish  morphologically  or  cul- 
turally from  the  true  dysentery  bacillus,  but  which  showed  differences  in 
their  serum  reaction.  By  careful  study  of  the.  behavior  of  these  bacilli 
in  the  serum  of  patients  and  in  immune  serum  from  animals,  he  not 
only  showed  that  they  were  different  from  his  original  cultures  from 
cases  of  epidemic  dysentery  which,  no  matter  what  their  source,  were 
found  to  be  alike,  but  that  they  showed  differences  among  themselves 
and  apparently  fell  into  two  or  more  varieties.  One  of  these  organisms 
culturally  and  by  its  serum  reactions  showed  itself  practically  identical 
with  one  of  the  cultures  he  had  received  from  Flexner. 

Spronck  ^  in  1901  described  an  organism  isolated  in  Utrecht  from 
dysentery  cases,  which  showed  great  similarity  to  the  Shiga-Kruse 
organism;  but,  when  tested  in  the  serum  of  a  horse  immunized  against 
true  dysentery  bacillus,  showed  practically  no  agglutination.  He  placed 
this  organism  in  the  group  designated  by  Kruse  as  the  '^  pseudo-dysentery 
bacilli."  His  communication  is  of  importance,  since  it  is  the  first  re- 
ported instance  in  which  any  investigator  had  recognized  and  associated 
the  so-called  pseudo-dysentery  bacilli  with  dysentery  approaching  the 
acute  epidemic  form  in  type. 

Following  this  work  a  number  of  investigators,  including  Vedder 

^  Kruse,  Deut.  med.  Woch.,  xxvi,  1900. 
'  Kruse,  Deut.  med.  Woch.,  xxvii,  1901. 
•/Sp'onc/c,  Ref,  Baumgarten's  Jahresber.,  1901. 


438  PATHOGENIC  MICROORGANISMS 

and  Duval/  Flexner,  and  Shiga  ^  himself,  published  communications  ir» 
which  they  claimed  identity  for  the  various  forms  previously  described. 

In  1902  Park  ^  and  Dunham  described  an  organism  which  they 
found  in  a  small  outbreak  of  dysentery  occurring  in  Maine.  This 
organism  differed  from  most  of  those  previously  described  in  that  it 
was  found  to  produce  indol  in  pepton  solutions. 

In  the  same  year  Martini  *  and  Lentz  published  an  article  in  which 
they  attempted  to  differentiate  various  dysentery  bacilli  by  means  of 
agglutination.  This  research  is  of  importance  in  that  it  supported  the 
work  of  Kruse  and  of  Spronck,  indicating  a  difference  between  the  ag- 
glutinative character  of  the  Kruse  organism  and  the  so-called  "  pseudo- 
dysentery"  type,  in  which  Flexner's  organisms  were  included.  It  is  of 
further  interest,  since  it  indicated  a  marked  difference  between  Flexner's 
Philippine  cultures  and  the  Philippine  culture  of  Strong,  the  Strong 
organism  refusing  to  agglutinate  not  only  in  ^' Shiga''  immune  serum, 
but  also  in  "  Flexner "  immune  serum. 

Simultaneously  with  this  article  Lentz  ^  published  the  results  of  com- 
parative cultural  researches  with  dysentery  and  "pseudo-dysentery" 
bacilli,  in  which  he  made  the  important  observation  that  the  original 
Shiga-Kruse  bacilli  did  not  affect  mannit,  while  the  "  pseudo-dysentery  " 
bacilli,  including  Flexner's  and  Strong's  Philippine  cultures,  fermented 
mannit,  giving  rise  to  a  distinct  acid  reaction  in  the  medium.  The 
Flexner  organisms  and  others  of  the  "pseudo-dysentery"  bacilli,  how- 
ever, fermented  maltose,  while  the  Shiga-Kruse  type,  as  well  as  Strong's 
bacillus,  left  it  unchanged  at  the  end  of  forty-eight  hours. 

In  January,  1903,  Hiss  and  Russell  ®  described  a  bacillus  ("  Y")  from 
a  case  of  fatal  diarrhea  in  a  child,  which  by  ordinary  cultural  test  and 
absence  of  motility  was  found  to  resemble  the  Shiga-Kruse  and  Flexner 
bacilli.  Immediately  upon  its  isolation,  it  was  found,  however,  to  differ 
from  the  Kruse  culture  by  its  ability  to  ferment  mannit.  This  observa- 
tion was  made  independently  of  Lentz's  work,  which,  at  that  time,  had 
not  become  known  in  America.  In  the  comparative  study  of  Hiss  and 
Russell  on  the  fermentative  abiHties  of  various  dysentery  cultures,  the 
serum  water  media  (described  on  page  132).  were  used.     By  the  use  of 

'  Vedder  and  Duval,  Jour,  Exp.  Med.,  vi,  1902. 

^  Shiga,  Zeit.  f.  Hyg.,  41,  1902. 

«  Park  and  Dunham,  N.  Y.  Univ.  Bull,  of  Med.  Sci.,  1902. 

*  Martini  und  Lentz,  Zeit.  f.  Hyg.,  xli,  1902. 

» Lentz,  Zeit.  f.  Hyg.,  xli,  1902. 

•Hiss  and  Russell  Med.  News,  Feb.,  1903. 


THE  DYSENTERY  BACILLI  439 

these  media,  it  was  found  that  the  Kruse  culture,  a  culture  of  Flexner's 
bacillus  from  the  Philippines,  and  Duval's  "  New  Haven  '*  culture  fer- 
mented dextrose  with  the  production  of  a  solid  acid  coagulum,  but  did 
not  affect  mannit,  maltose,  saccharose,  or  dextrin.  The  culture  of  Hiss 
and  Russell,  on  the  other  hand,  fermented  not  only  dextrose  but  also 
mannit  with  the  production  of  acid  and  coagulation  of  the  medium. 
Maltose,  saccharose,  and  dextrin  were  not  fermented.  The  "  Y  "  bacillus, 
furthermore,  was  shown  to  differ  entirely  from  the  cultures  of  Shiga, 
Kruse,  and  "New  Haven"  in  the  serum  of  immunized  animals.  This 
serum  had  for  bacillus  "  Y  "  a  titer  of  1  :  500  while  the  three  other  above- 
named  organisms  did  not  agglutinate  in  it  at  any  dilution.  In  normal 
beef  serum,  the  Hiss-Russell  organism  was  found  to  agglutinate  as  highly 
at  1  :  320,  while  the  other  three  cultures  gave  no  reaction  in  dilutions  of 
over  1  :  10  or  20. 

Park  and  Carey/  in  March,  1903,  described  an  epidemic  of  dysen- 
tery occurring  in  the  town  of  Tuckahoe,  near  New  York  City,  and 
isolated  an  organism  which  resembled  the  Shiga-Kruse  bacilli  in  not 
fermenting  mannit,  but  produced  indol  in  pepton  solution  after  five 
days.  It  corresponded  in  agglutination  with  the  cultures  "  New  Haven '' 
and  "Shiga"  when  tested  in  the  serum  of  a  goat  immunized  against 
the  mannit-fermenting  culture  "Baltimore,"  i.e.,  did  not  react  at  1:50, 
whereas  Flexner's  "  Manila  "  and  "  Baltimore  "  cultures.  Park  and  Dun- 
ham's "Seal  Harbor"  culture,  and  some  New  York  cultures,  all  fer- 
menting mannit,  agglutinated  up  to  two  thousand  dilution  in  the  "  Bal- 
timore" serum. 

The  preceding  review  of  a  part  of  the  literature,  by  which  our  knowl- 
edge of  the  dysentery  bacilli  was  developed,  demonstrates  sufficiently 
that  we  have  to  deal  in  this  group  with  a  number  of  different  micro- 
organisms. This,  as  we  have  seen,  was  a  fact  first  recognized  by  Kruse 
when  he  spoke  of  his  true  dysentery  and  his  pseudo-dysentery  strains. 
In  spite  of  much  confusion  at  first,  the  careful  study  of  fermentation 
phenomena,  of  specific,  agglutinations,  and,  more  recently,  by  Ohno  ^ 
and  others,  of  the  bacteriolytic  phenomena  in  immune  sera,  has  made 
it  possible  to  distinguish  sharply  between  a  number  of  groups. 

Basing  the  grouping  of  these  microorganisms  upon  a  careful  study 
of  fermentations,  Hiss^  has  divided  them  as  follows: 


»  Park  and  Carey,  Jour,  Med'  Res.,  ix,  1903. 
» Ohno,  Philippine  Jour,  of  Sci.,  1,  ix.,  1906. 
*Hiss,  Jour.  Med.  Res.,  N.  S.,  viii,  1904. 


440  PATHOGENIC  MICROORGANISMS 


"Kruse,"  >      Ferment  dextrose.     Group  I. 

"New  Haven" 


"Y"  (Hiss  and  Russell  type) 

"SealHarbbr" 

"  Diamond  " 

"Ferra" 

"Strong"  (type) 

"  Harris  "  (type) 
"Gray" 
"Baltimore" 
"Wollstein" 


Ferment  dextrose  and  mannit.    Group  II. 


Ferments      dextrose,     mannit,     saccharose 
Group  III. 

Ferment  dextrose,  mannit,  maltose,  saccha- 
rose, dextrin.  Fermentation  of  saccharose 
(as  a  rule)  only  after  6  days.    Group  IV. 


It  was  noticed,  it  should  be  mentioned,  however,  that  in  the  case  of  the 
a  Y  "  "Diamond,"  and  "Ferra"  there  was  usually  delayed  acid  fermen- 
tation of  maltose,  never  any  of  dextrin. 

In  studying  the  agglutinative  characters  of  these  groups,  furthermore, 
it  was  found  that  fermentation  tests  and  agglutinations  went  hand  in 
hand.    The  following  table  will  illustrate  this  point:/    ' 

Serum  of  Rabbit  immunized  against  GRobp  I.  (Shiga's  culture). 

Bacilli  of  Group  I. : 

"  Shiga  "  (homologous) 20,000 

"Kruse" .............;..!'..  20,000 

"New  Haven" •. 20,000 

Bacilli  of  Group  II.: 

"Y" 200 

"Ferra" ..-..  200 

"Seal  Harbor" 200 

Bacilli  of  Group.  IV.: 

"Baltimore" 800 

"Harris" 800 

"Gray" ...,....,......,.................,..>  800 

"Wollstein"  ......  ^  ......!  . ...  ..........  .^  . . . . ....,..,  800 

Serum  of  Rabbit  immunized  against  Group  II.  ("Y"  culture, 
Hiss  and  Russell). 

Bacilli  of  Group  I.:  ^ 

"Shiga" less  than  100 

"Kruse" 100 

"New  Haven"  .. . . . .' 100 

1  Hiss,  Jour,  of  Med.  Research,  13,  N.  S.",  viii,  190i^. 


THE  DYSENTERY  BACILLI  441 

Bacilli  of  Group  II. : 

"  Y "  (homologous)    6,400 

"Ferra" 6,400 

"Seal  Harbor 6,400 

Bacilli  of  Group  IV.: 

"Baltimore  " 1,600 

"Gray" 1,600 

"Harris" 1,600 

"WoUstein" 1,600 

Serum  of  Rabbit  immunized  against  Group  IV.  ("Baltimore" 
culture) . 

Bacilli  of  Group  I.: 

"Shiga"    less  than  100 

"Kruse"  100 

"New  Haven " 100 

Bacilli  of  Group  II. : 

"  Y" 400 

"Ferra" 400 

"Seal  Harbor" 400 

BaciUi  of  Group  IV.: 

.    "Baltimore"  (homologous) ,. .  3,200 

"Harris" 3,200 

"Gray" 3,200 

"WoUstein"  3,200 

In  common,  all  these  groups  possess  an  identical  morphology,  the 
Gram-negative  staining  characteristics,  the  lack  of  motility  with  close 
adherence  to  the  line  of  inoculation  in  the  Hiss  tube  medium,  the  in- 
ability to  liquefy  gelatin,  the  inability  to  form  acid  from  lactose,  and 
the  inability  to  produce  gas  from  any  carbohydrate  media. 

Biological  Considerations. — The  dysentery  bacilli  in  neutral  broth 
or  upon  agar  slants  may  remain  alive  without  transplantation  for 
periods  of  several  months.  They  are  aerobes  and  facultative  anaerobes 
when  proper  sugars  are  present,  preferring,  however,  the  aerobic  environ- 
ment. They  are  easily  destroyed  by  heat,  an  exposure  to  60°  C.  killing 
them  usually  in  a  short  time  (ten  minutes).  Against  cold  they  show 
considerable  resistance,  surviving  freezing  for  a  period  of  several  weeks. 
They  show  little  resistance  to  the  usual  strengths  of  the  common  chem- 
ical disinfectants. 

Pathogenicity. — There  is  practically  no  doubt  at  the  present  time  as  to 
the  etiological  connection  between  the  bacilli  of  this  group  and  the  dis- 
eases clinically  classified  as  acute  dysentery.  A  more  chronic  form  of 
29 


442  PATHOGENIC   MICROORGANISMS 

dysentery  due  to  a  protozoan,  the  Amoeba  coli,  though  presenting  much 
clinical  resemblance  to  the  bacillary  dysenteries  is,  nevertheless,  an 
entirely  distinct  disease. 

Infection  takes  place,  probably,  entirely  by  ingestion  of  the  bacteria 
with  infected  water  or  food  contaminated  from  the  feces  of  dysentery 
patients.  A  small  epidemic  occurring  in  a  hospital  in  New  York  City 
and  caused  by  the  bacillus  ^'Y"  of  Hiss  and  Russell  was  indirectly 
traced  to  milk  by  Zinsser.^ 

Endemic  in  a  large  part  of  the  world,  especially  in  the  warmer 
climates,  the  disease  most  frequently  occurs  in  epidemics  of  more  or 
less  definite  localization,  usually  under  conditions  which  accompany 
the  massing  of  a  large  number  of  human  beings  in  one  place,  such  as 
those  which  occur  in  the  crowded  quarters  of  unsanitary  towns,  in  insti- 
tutions such  as  insane  asylums,  or  in  military  camps.  The  mortality  of 
such  epidemics  may  be  very  large.  According  to  Shiga,^  the  disease  in 
Japan  frequently  shows  a  mortality  of  over  twenty  per  cent. 

The  disease  in  human  beings  usually  begins  as  an  acute  gastro- 
enteritis which  is  accompanied  by  abdominal  pain  and  diarrhea.  As 
it  becomes  more  severe,  the  colicky  pains  and  diarrhea  increase,  the 
stools  lose  their  fecal  character,  becoming  small  in  quantity  and  filled 
with  mucus  and  flakes  of  blood.  There  is  often  severe  tenesmus  at 
this  stage,  and  the  bacilli  are  present  in  large  numbers  in  the  dejecta. 
Owing  to  the  absorption  of  toxic  products,  symptoms  referable  to  the 
nervous  system,  such  as  muscular  twitching,  may  supervene,  and  if  the 
disease  is  at  all  prolonged,  there  are  marked  inanition  and  prostration. 

At  autopsy  in  early  stages  there  may  be  found  only  a  severe  catar- 
rhal inflammation  of  the  mucous  membrane  of  the  large  intestine.  In 
the  later  stages  there  are  extensive  ulcerations,  and  the  bacteria  are 
histologically  found  lodged  within  the  depths  of  the  mucosa  and  sub- 
mucosa.  Occasionally  they  may  penetrate  to  the  mesenteric  glands,  but 
as  far  as  we  know  there  is  no  penetration  into  the  general  circulation. 

Poisonous  Products  of  the  Dysentery  Bacilli. — The  separate  types  of 
dysentery  bacilli  vary  exceedingly  in  their  powers  to  pro  luce  toxic 
substances.  Of  all  the  various  types  which  have  been  described,  the 
strongest  poisons  have  been  produced  with  baciUi  of  the  Shiga-Kruse 
variety,  less  regularly  active  ones  with  bacilli  of  the  Flexner  and  of  the 
"Y"  type.  In  fact,  investigations  carried  out  with  the  Shiga  bacillus 
have  tended  to  show  that  the  disease  itself  is  probably  a  true  toxemia, 

^Zinsser,  Proc.  N.  Y.  Path.  Soc,  1907      2  Shiga.  Cent.  f.  Bakt.,  xxiii.  1898. 


THE  DYSENTERY  BACILLI  443 

its  symptoms  being  referable  almost  entirely  to  the  absorption  of  the 
poisonous  products  of  the  bacillus  from  the  intestine. 

The  earliest  investigations,  carried  on  chiefly  upon  rabbits,  which 
are  more  suscjptible  to  this  poison  than  any  other  animals,  showed  that 
even  small  doses  of  cultures  of  this  bacillus  administered  intravenously 
or  subcutaneously  would  produce  death  within  a  very  short  time. 
Conradi,^  Vaillard^  and  Dopter,  and  others,  finding  that  toxic  symptoms 
were  almost  as  pronounced  when  dead  cultures  were  given  as  when  the 
living  bacilli  were  administered,  came  to  the  conclusion  that  the  poisons 
of  this  bacillus  were  chiefly  of  the  endotoxin  type.  More  recently  Todd,^ 
Kraus,''  and  Rosenthal^  have  claimed  independently  that  they  were 
able  to  demonstrate  strong  soluble  toxins,  similar  in  every  way  to  diph- 
theria toxin.  Kraus  and  Doerr,^  moreover,  claim  to  have  further  cor- 
roborated this  by  producing  specific  antitoxins  with  these  substances. 

It  is  easy  to  obtain  poisonous  substances  from  dysentery  cultures 
in  considerable  strength,  both  by  extracting  the  bacilli  themselves 
and  by  filtration  of  properly  prepared  cultures.  It  is  therefore  not 
unlikely  that  both  types  of  poison  are  produced  by  the  bacilli. 
Neisser  and  Shiga  ^  obtained  toxins  by  emulsifying  agar  cultures  in 
sterile  salt  solution,  killing  the  bacilli  at  60°  C,  and  allowing  them  to 
extract  at  37.5°  C.  for  three  days  or  more.  The  filtrates  from  such  emul- 
sions were  extremely  toxic.  The  simplest  method  of  obtaining  poisons 
from  these  bacilli  is  to  cultivate  them  for  a  week  or  longer  upon  moder- 
ately alkahne  meat-infusion  broth.  At  the  end  of  this  time,  the  micro- 
organisms themselves  may  be  killed  by  heating  to  60°  and  the  cultures 
filtered.  According  to  Doerr,^  the  toxins  may  be  obtained  in  the  dry 
state  by  precipitation  with  ammonium  sulphate  and  re-solution  of  the 
precipitate  in  water. 

The  action  of  th.e  dysentery  toxin  upon  animals  is  extremely 
characteristic  and  throws  much  light  upon  the  disease  in  man.  The 
injection  of  a  large  dose  intravenously  into  rabbits  causes  a  rapid 
fall  in  temperature,  marked  respiratory  embarrassment,  and  a  violent 


1  Conradi,  Deut.  med.  Woch.,  1903, 

2  Vaillard  et  Dopter,  Ann,  de  Tinst,  Pasteur,  1903. 

3  Todd,  Brit.  Med,  Jour,,  Dec,  1903,  and  Jour,  of  Hyg,,  4,  1904. 
^  Kraus,  Monatschr,  f.  Gesundheit,  Suppl.  11,  1904. 
»/2osen^/iaZ,  Deut,  med.  Woch.,    1904. 

*  Kraus  und  Doerr,  Wien.  klin.  Woch,,  xlii,  1905. 

7  Neisser  and  Shiga,  Deut.  med,  Woch.,  1903. 

8  Doerr,  "  Das  Dysenterietoxin,"  Jena,  1907. 


444  PATHOGENIC   MICROORGANISMS 

diarrhea.  This  is  at  first  watery,  later  contains  large  amounts  of  blood. 
If  the  animals  live  a  sufficient  length  of  time,  paralysis  may  occur,  the 
animal  may  fall  to  one  side  or  may  drag  its  posterior  extremities.  It  is 
a  remarkable  fact  that  intravenous  inoculation  gives  rise  to  intestinal 
inflammation  of  a  severe  nature,  unquestionably  due  to  the  excretion 
of  the  poison  by  the  intestinal  mucosa  and  limited,  usually,  to  the  ce- 
cum and  colon,  rarely  attacking  the  small  intestine.  Flexner,^  who  has 
experimented  extensively  upon  this  question,  believes  it  probable  that 
most  of  the  pathological  lesions  occurring  in  the  intestinal  canal  of  dysen- 
tery patients  are  referable  to  this  excretion  of  dysentery  toxin,  rather 
than  to  the  direct  local  action  of  the  bacilli. 

Toxins  from  the  Shiga-Kruse  type  are  the  most  potent  and  those 
which  cause  paralysis. 

Immunization  with  Dysentery  Bacilli. — The  immunization  of  small 
animals,  such  as  rabbits  and  guinea-pigs,  against  dysentery  bacilli, 
especially  those  of  the  Shiga  type,  is  attended  with  much  difficulty, 
owing  to  the  great  toxicity  of  the  cultures.  Nevertheless,  successful 
results  may  be  accomplished  by  the  administration  of  extremely  small 
doses  of  living  or  dead  bacilli,  increased  very  gradually  and  at  sufficient 
intervals.  Horses  may  be  more  easily  immunized.  The  serum  of  such 
actively  immunized  animals  contains  agglutinins  in  considerable  con- 
centration and  of  a  specificity  sufficiently  illustrated  in  the  preceding 
section  dealing  with  the  identification  of  the  various  species.  For 
diagnostic  purposes  in  human  beings,  the  agglutination  reaction,  accord- 
ing to  the  technique  of  the  Widal  reaction  for  typhoid  fever,  has  been 
utilized  by  Kruse  ^  and  others.  According  to  most  observers,  normal 
\uman  serum  never  agglutinates  dysentery  bacilli  in  dilutions  greater 
than  one  in  twenty,  while  the  serum  of  dysentery  patients  will  often  be 
active  in  dilutions  as  high  as  one  in  fifty. 

Bactericidal  substances  have  been  demonstrated  in  the  serum  of  im- 
munized animals  as  well  as  in  the  serum  of  diseased  human  beings. 
These  have  been  determined,  in  vitro,  by  Shiga,^  and  by  the  intraperito- 
neal technique  of  Pfeiffer  by  Kruse."*  Bacteriolysis  may  take  place  in 
high  dilutions  of  the  serum,  and  has  recently  been  used  for  the  differen- 
tiation of  the  types  of  the  dysentery  bacilli  by  Ohno.^ 

True  antitoxins  in  immune  sera  have  been  recently  described  by 
Kraus  and  Doerr,* 

»  Flexner,  Jour.  Exp.  Med.,  8,  1906.  2  Kruse,  Deut.  med.  Woch.,  1901. 

3  Shiga,  Zeit.  f.  Hyg.,  xli.  *  Kruse,  Deut.  med.  Woch.,  1903, 

«  Ohno,  Philippine  Jour,  of  Sci.,  vol.  i,  1906.    «  Kraus  und  Doerr,  loc.  cit. 


THE    DYSENTERY    BACILLI 


445 


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446  PATHOGENIC  MICROORGANISMS 

Passive  immunization  of  animals  and  human  beings  with  the  serum 
of  highly  immunized  horses  has  been  variously  attempted  by  Shiga/ 
Kraus/  Gay/  and  others.  All  these  observers  have  reported  distinct 
benefit  to  the  patients  and  a  reduction  of  the  mortality  by  the  use  of 
such  sera.  Striking  and  rapid  reductions  of  temperature  and  rapid  con- 
valescence, after  a  sjngie  injection,  have  occasionally  been  observed. 
The  earlier  workers  were  inclined  to  attribute  the  beneficial  results  of 
these  sera  entirely  to  their  bactericidal  value. 

Todd  has  recently  demonstrated  that  the  mixture  of  such  an  immune 
serum  with  solutions  of  toxin  and  exposure  of  the  mixture  at  37.5°  C. 
for  a  half  hour  would  produce  almost  complete  neutralization  of  the 
poison,  thus  demonstrating  that  at  least  a  large  part  of  the  beneficial 
action  of  the  immune  sera  was  due  to  a  true  antitoxic  process.  Be- 
cause of  the  different  varieties  of  dysentery  bacilli,  polyvalent  serum  has 
been  recommended.  Prophylactic  vaccination  of  human  begins  with 
dead  dysentery  cultures  has,  so  far,  led  to  no  practical  result. 

Shiga,  Deut.  med.  Woch.,  1901,  ^  Kraus,  loc.  cit. 

8  Gay,  Penn.  Med.  Bull.,  1902. 


CHAPTER  XXX 

BACILLUS    MUCOSUS   CAPSULATUS,    BACILLUS    LACTIS    AEROGENESj 

BACILLUS    PROTEUS 

BACILLUS    MUCOSUS   CAPSULATUS 

(Bacterium  pneumonice,  Friedldnder' s  bacillus,  Pneumohacillus) 

In  1882,  Friedlander  ^  announced  the  discovery  of  a  microorganism 
which  he  believed  to  be  the  incitant  of  lobar  pneumonia  and  which,  in 
his  original  communications,  he  described  as  a  "  micrococcus." 

A  superficial  morphological  resemblance  between  Friedlander's 
microorganism  and  Diplococcus  lanceolatus,  now  recognized  as  the  most 
frqquent  cause  of  lobar  pneumonia,  led,  at  first,  to  much  confusion,  and 
it  was  not  until  several  years  later,  owing  to  the  careful  researches  of 
Frankel  ^  and  of  Weichselbaum,^  that  the  "  micrococcus  "  of  Friedlander 
was  recognized  as  a  short,  encapsulated  bacillus  which  occurred  in 
lobar  pneumonia  exceptionally  only.  Similar  bacilli  were  subsequently 
found  by  other  observers,  bacilli  which,  mainly  upon  morphological 
grounds,  are  classified  together  as  the  ^'Friedlander  group,''  or  the 
''group  of  Bacillus  mUcosus  capsulatus." 

Morphology  and  Staining. — The  Friedlander  bacillus  is  a  short,  plump 
bacillus  with  rounded  ends,  subject  to  great  individual  variations  as  to 
size.  Its  average  measurements  are  from  0.5  to  1.5  micra  in  width  and 
0.6  to  5  micra  in  length.  Forms  approaching  both  extremes  may  be  met 
with  in  one  and  the  same  culture.  The  short,  thick  forms,  frequently 
found  in  animal  and  human  lesions,  are  almost  coccoid  and  account  for 
Friedlander's  error  in  first  describing  the  bacillus  as  a  tnicrococcus. 
The  bacilli  may  be  single,  in  diplo-form,  or  in  short  chains.  They  are 
non-motile  and  possess  no  flagella.    Spores  are  not  formed. 

The  bacillus  is  characteristically  surrounded  by  a  well-developed 
capsule  which  is  most  perfectly  demonstrated  in  preparations  taken 
directly  from  some  animal  fluid,  such  as  the  secretion  or  exudate 
from  infected  areas.    It  is  also  seen,  however,  in  smears  made  from  agar 

^Friedlander,  Virchow's  Arch.,  Ixxxvii,  1882;  Fort.  d.  Med.,  i,  1883;  ibid., 
ii.  1884. 

2  Frankel,  Zeit.  f.  klin.  Med.,  x,  1886. 
•  Weichselbaum,  Med.  Jahrb.,  Wien,  1886. 

447 


448  ^  PATHOGENIC  MICROORGANISMS" 

or  gelatin  cultures.  The  capsule  is  usually  large,  twice  or  three  times 
the  size  of  the  bacillus  itself.  When  seen  in  chains  or  in  groups,  several 
bacilli  may  appear  to  be  inclosed  in  one  capsule.  Prolonged  cultivation 
on  agar  or  gelatin  may  result  in  disappearance  of  the  capsule.  The  bacil- 
lus is  easily  stained  with  the  ordinary  dyes,  but  is  decolorized  when 
stained  by  the  Gram-method.  Capsules  may  often  be  seen  when  the 
more  intense  anilin  dyes  are  employed.  They  are  brought  out  with  much 
regularity  by  any  of  the  usual  capsule  stains. 

Cultivation. — B.  mucosus  capsulatus  is  easily  cultivated.    It  grows 


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Fig.  94. — Bacillus  mlcosus  capsulatus. 

readily  on  all  the  usual  culture  media,  both  on  those  haying  a  meat- 
infusion  basis  and  on  those  made  with  meat  extract.  Growth  takes 
place  at  room  temperature  (18°  to  20°)  and  more  rapidly  at  37.5°  C.  A 
temperature  of  60°  C.  and  over  kills  the  bacilli  in  a  short  time.  The  ther- 
mal death-point  according  to  Sternberg  is  56°  C.  Growth  ceases  below 
10°  to  12°  C.  Kept  at  room  temperature  and  protected  from  drying, 
the  bacillus  may  remain  alive,  in  cultures,  for  several  months. 

The  bacillus  is  not  very  fastidious  as  to  reaction  of  media,  growing 


BACILLUS  MUCOSUS  CAPSULATUS  449 

equally  well  on  moderately  alkaline  or  acid  media.  It  is  aerobic  and 
facultatively  anaerobic;  growth  under  anaerobic  conditions,  however, 
is  not  luxuriant. 

On  agar,  growth  appears  in  the  form  of  grayish-white  mucus-hke 
colonies,  having  a  characteristically  slimy  and  semi-fluid  appearance. 
Colonies  have  a  tendency  to  confluence,  so  that  on  plates,  after  three  or 
four  days,  a  large  part  of  the  surface  appears  as  if  covered  with  a  film  of 
glistening,  sticky  exudate,  which,  if  fished,  comes  off  in  a  tenacious, 
stringy  manner.  It  is  often  possible  to  make  a  tentative  diagnosis  of 
the  bacillus  from  the  appearance  of  this  growth. 

In  broth,  there  is  rapid  and  abundant  growth,  with  the  formation 
of  a  pellicle,  general  clouding,  and  later  the  development  of  a  profuse, 
stringy  sediment. 

Stab  cultures  in  gelatin  show,  at  first,  a  white,  thin  line  of  growth 
along  the  course  of  the  puncture.  .Soon,  however,  rapid  growth  at 
the  top  results  in  the  formation  of  a  grayish  mucoid  droplet  on  the 
surface,  which,  enlarging,  gives  the  growth  a  nail-like  appearance.  This 
nail-shape  was  originally  described  by  Friedlander  and  regarded  as  diag- 
nostic for  the  bacillus.  The  gelatin  is  not  fluidified.  As  the  culture  grows 
older  the  entire  surface  of  the  gelatin  tube  may  be  covered  with  growth, 
flowing  out  from  the  edges  of  the  nail-head.  The  gelatin  acquires  a  darker 
color  and  there  may  be  a  few  gas  bubbles  below  the  surface.  Micro- 
scopically, colonies  on  gelatin  plates  have  a  smooth  outline  and  a  finely 
granular  or  even  homogeneous  consistency. 

On  blood  serum,  a  confluent  mucus-like  growth  appears. 

On  'potato,  abundant  growth  appears,  slightly  more  brownish  in  color 
than  that  on  other  media. 

In  pepton  solutions,  there  is  no  indol  formation. 

In  milk,  there  is  abundant  growth  and  marked  capsule  develop- 
ment.    Coagulation  occurs  irregularly. 

In  considering  the  general  cultural  characteristics  of  the  Fried- 
lander  bacillus,  it  must  hot  be  forgotten  that  we  are  dealing  with  a 
rather  heterogeneous  group,  the  individuals  of  which  are  subject  to 
many  minor  variations.  Capsule  development,  lack  of  motility,  in- 
ability to  fluidify  gelatin,  failure  to  form  indol,  and  absence  of  spores,  are 
characteristics  common  to  all.  In  size,  general  appearance,  gas  forma- 
tion, and  pathogenicity,  individual  strains  may  vary  much,  one  from 
the  other.     Strong  ^  has  studied  various  races  as  to  gas  formation  and 

^  Strong,  Cent.  f.  Bakt.,  xxv,  1899. 


450  PATHOGENIC   MICROORGANISMS 

concludes  that  most  strains  form  gas  from  dextrose  and  levulose,  but 
that  lactose  is  fermented  by  some  only.  About  two-thirds  of  the  gas 
formed  is  hydrogen,  the  rest  CO2.  Acid  formation,  according  to  Strong, 
is  also  subject  to  much  variation  among  different  races.  Similar  studies 
by  Perkins  ^  show  that  most  of  the  ordinary  cultural  characteristics  of 
bacilli  of  this  group  are  extremely  variable  and  can  not  serve  as  a  basis 
for  differentiation.  Reactions  on  sugars,  however,  are  more  constant. 
Perkins  suggests  the  following  tentative  division  classes  on  this  basis: 

I.  All  carbohydrates  fermented  with  the  formation  of  gas. 

II.  All  carbohydrates,  except  lactose,  fermented  with  the  formation 
of  gas. 

III.  All  carbohydrates,  except  saccharose,  fermented  with  the 
formation  of  gas. 

Type  I.  corresponds  to  B.  aerogenes  (Migula),  Type  II.  to  B. 
Friedlander  or  Bacterium  pneumoniae  (Migula),  and  Type  III.  to 
Bacillus  lactis  aerogenes. 

Differentiation  by  means  of  sermn  reactions  has  not  proved  satis- 
factory. 2 

Pathogenicity. — When  Friedlander  first  described  this  microorganism, 
he  assumed  it  to  be  the  incitant  of  lobar  pneumonia.  Subsequent  re- 
searches by  Weichselbaum  ^  and  others  have  shown  it  to  be  etiologically 
associated  with  pneumonia  in  about  seven  or  eight  per  cent  of  all  cases. 
The  percentage  in  this  country  is  probably  lower.  Such  cases  can  often 
be  diagnosed  by  the  presence  of  the  bacilli  in  the  sputum,  which  is  pecul- 
iarly sticky  and  stringy.  Cases  of  Friedlander  pneimionia  are  extremely 
severe  and  usually  fatal.  The  bacillus  has  been  found  in  cases  of  ulcer- 
ative stomatitis  and  nasal  catarrh;  in  two  cases  of  severe  tonsillitis 


in  children;  in  the  pus  from  suppurations  in  the  antrum  of  Highmore 
and  the  nasal  sinuses  (Frankel  and  others),  and  in  cases  of  fetid  cory- 
za  (ozena),  of  which  disease  it  is  supposed  by  Abel^  and  others  to 
be  the  specific  cause.    Whether  the  ozena  bacillus  represents  a  separate 

1  Perkins,  Jour,  of  Infect.  Dis.,  I,  No.  2,  1904. 

2  J,  G.  Fitzgerald,  who  has  recently  made  a  careful  study  of  the  mucosus  cap- 
sulatus  group  has  concluded  that  present  methods  do  not  permit  a  subdivision  of 
these  organisms  into  separate  species.  He  offers  the  following  ' '  tentative  suggestion ' ' : 
It  is  conceivable  that  mutations  based  on  the  necessity  of  maintaining  a  parasitic 
existence  have  caused  Gram-negative  badlli  found  normally  in  the  body  elsewhere 
than  in  the  intestinal  tract  to  develop  capsules  for  protection  and  a  new  group  has 
arisen  which  we  designate  B.  mucosus  capsulatus;  and  the  varieties  B.  aerogenes 
and  B.  acidi  lactici  connect  the  group  with  the  non-encapsulated  colon  group." 

3  Weichselbaum,  loc.  cit,  *  Abel,  Zeit.  f .  Hyg.,  xxi. 


BACILLUS  OF   RHINOSCLEROMA  451 

species  or  not,  can  not  at  present  be  decided.  The  bacillus  of  Fried- 
lander  has  been  found  in  empyema  fluid,  in  pericardial  exudate  (after 
pneumonia),  and  in  spinal  fluid. ^  Isolated  cases  of  Friedlander  bacillus 
septicemia  have  been  described. ^  Being  occasionally  a  saprophytic 
inhabitant  of  the  normal  intestine,  it  has  been  believed  to  be  etiologic- 
ally  associated  with  some  forms  of  diarrheal  enteritis. 

B.  mucosus  capsulatus  is  pathogenic  for  mice  and  guinea-pigs,  less  so 
for  rabbits.  Inoculation  of  susceptible  animals  is  followed  by  local  in- 
flammation and  death  by  septicemia.  If  inoculation  is  intraperitoneal, 
there  is  formed  a  characteristically  mucoid,  stringy  exudate. 

The  question  of  immunization  against  bacilU  of  the  Friedlander 
group  is  still  in  the  stage  of  experimentation.  Immunization  with  care- 
fully graded  doses  of  dead  bacilh  has  been  successful  in  isolated  cases. 
Specific  agglutinins  in  immune  serum  have  been  found  by  Clairmont,^ 
but  irregularly  and  potent  only  against  the  particular  strain  used  for 
the  immunization. 

OTHER    BACILLI    OF    THE    FRIEDLANDER    GROUP 

Bacillus  of  Rhinoscleroma. — This  bacillus,  discovered  by  v.  Frisch  * 
in  1882,  is  a  plump,  short  rod,  with  rounded  ends,  morphologically 
almost  identical  with  Friedlander's  bacillus;  it  is  non-motile  and  pos- 
sesses a  distinct  capsule.  Although  at  first  described  as  Gram-positive, 
it  has  been  shown  to  be  decolorized  with  this  method  of  staining.  Cul- 
turally it  is  almost  identical  with  B.  mucosus  capsulatus.  It  forms 
slimy  colonies,  has  a  nail-like  appearance  in  gelatin  stab  cultures,  and  in 
pepton  solutions  produces  no  indol.  It  differs  from  B.  mucosus  cap- 
sulatus (Wilde  ^)  in  forming  no  gas  in  dextrose  bouillon,  in  producing 
no  acid  in  lactose  bouillon,  and  in  never  coagulating  milk. 

Pathogenicity. — The  bacillus  of  rhinoscleroma  is  but  moderately 
pathogenic  for  animals  delicately  susceptible  to  the  bacillus  of  Fried- 
lander. Rhinoscleroma,  the  disease  produced  by  this  bacillus  in  man, 
consists  of  a  slowly  growing  granulomatous  inflammation,  located  usu- 
ally at  the  external  nares  or  upon  the  mucosa  of  the  nose,  mouth, 
pharynx,  or  larynx.  It  is  composed  of  a  number  of  chronic,  hard, 
nodular  swellings,  which,  on  histological  examination,  show  granulation 
tissue  and  productive  inflammation.     In  the  meshes  of  the  abundant 

1  Jdger,  Zeit.  f.  Hyg.,  xix.  ^  Howard,  Johns  Hopkins  Hosp.  Bull.,  1899. 

'  Clairmont,  Zeit.  f.  Hyg.,  xxxix.        ^  v.  Frisch,  Wien.  med.  Woch.,  1882. 
6  Wilde,  Cent.  f.  Bakt.,  xx,  1896. 


452 


PATHOGENIC  MICROORGANISMS 


connective  tissue  lie  many  large  swollen  cells,  the  so-called  "Mikulicz 
cells."  1  The  rhinoscleroma  baciUi  He  within  these  cells  and  in  the 
intercellular  spaces.  They  can  be  demonstrated  in  histological  sections 
and  can  be  cultivated  from  the  lesions,  usually  in  pure  culture.  Rhino- 
scleroma  is  rare  in  America.  It  is  most  prevalent  in  Southeastern 
Europe.  The  disease  is  slowly  progressive  and  comparatively  intract- 
able to  surgical  treatment, 
but  hardly  ever  affects 
the  general  health  unless 
by  mechanical  obstruction 
of  the  air  passages. 

B.  Ozaense. — The  work 
of  Abel  2  and  others  has 
shown  that  ozena,  or  fetid 
nasal  catarrh,  is  almost  al- 
ways associated  with  a 
bacillus  morphologically 
and  culturally  almost  iden- 
tical with  B.  mucosus 
capsulatus.  The  bacillus 
can  not  be  definitely  sepa- 
rated from  the  latter.  Ac- 
cording to  Wilde  ^  it  forms 
no  gas  in  dextrose  bouillon 
and  is  less  pathogenic  for 
mice  than  B.  Friedlander. 
Whether  it  is  a  separate 
species,  or  merely  an  atypical  form  changed  by  environment,  can  not 
be  stated  at  present. 

Perez  Bacillus  of  Ozsena. — Perez  ^  in  1899  described  another  micro- 
organism which  he  connects  etiologically  with  ozaena.  The  Perez  bacillus 
is  Gram-negative,  pleomorphic,  non-motile  and  non-capsulated.  It  grows 
easily  on  ordinary  media,  does  not  liquefy  gelatin,  and  makes  indol.  Its 
cultures  have  a  characteristic  fetid  odor.  Intravenously  injected  into 
rabbits  it  seems  to  produce  a  localized  lesion  in  the  nasal  cavity  on  the 
turbinated  bones.  Hofer  ^  has  also  isolated  it,  but  recent  work  leaves 
its  importance  as  the  causative  agent  in  doubt. 

1  Mikulicz,  Arch,  f .  Chir.,  xx,  1876.       2  ^5^;^  Zgit.  f.  Hyg.,  xxi.       ^  Wilde,  loc.  cit. 

*  Perez,  Animal  de  I'Inst.  Past.  1899. 

•^  Hofer,  Wien.  klin.  Woch.,  vol.  26,  pp.  1011  and  1628. 


Fig.  95. — Bacillus  of  Rhinoscleroma.  Sec- 
tion of  tissue  showing  the  microorganisms 
within  MikuUcz  cells.  (After  Frankel  and 
Pfeiffer.) 


BACILLUS  LACTIS  AEROGENES 


453 


BACILLUS  LACTIS  AEROGENES 

Bacillus  lactis  aerogenes  is  the  type  of  a  group  which  is  closely 
similar  to  the  colon  group  and  often  distinguished  from  it  with  difficulty. 
It  was  first  described  by  Escherich  ^  in  1885  who  isolated  it  from  the 
feces  of  infants.  Since  then  it  has  been  learned  that  this  bacillus  is  almost 
constantly  present  in  milk,  and,  together  with  one  or  two  other  micro- 
organisms, is  the  chief  cause  of  the  ordinary  souring  of  milk.  Apart  from 
its  occurrence  in  milk,  moreover,  the  bacillus  is  widely  distributed  in 
nature,  being  found  in  feces,  in  water,  and  in  sewage.  It  is  distinguish- 
able from  the  colon  bacillus  chiefly  by  the  fact  that  it  is  less  motile, 
possesses  no  flagella,  hardly  ever  forms  chains,  and,  when  cultivated  upon 
suitable  media,  especially  milk,  it  possesses  a  distinct  capsule.  It  differs 
from  the  colon  bacillus,  furthermore,  in  that  it  is  capable  of  fermenting 
polysaccharids,  such  as  starch,  and  does  not  form  indol  upon  pep- 
ton  media.  It  is  distinguishable  from  the  bacillus  of  Friedlander  (B. 
mucosus  capsulatus),  according  to  Wilde,^  by  its  more  energetic  gas 
formation  in  dextrose  broth,  its  abiUty  to  produce  acid  on  lactose  media, 
and  its  invariable  coagulation  of  milk.  Unlike  the  colon  bacilli,  it  does 
not  form  gas  on  Dulcit.^  It  differs  from  the  other  important  non-duldt 
fermentation,  the  bacillus  acidi  lactici,  in  fermenting  saccharose.  Ten- 
tative differentiations  of  these  bacilli  may  be  made  as  follows: 


Dextrose. 

Lactose. 

Saccharose. 

Dulcit. 

B.  coli  communis 

+ 
+ 
+ 

+ 
+ 
+ 
+ 

+ 

+ 

B.  coli  communior 

B.  lact.  aerogenes 

B.  lact.  acidi 

_ 

It  grows  upon  the  simplest  media,  is  a  facultative  anaerobe,  and 
grows  most  abundantly  at  a  temperature  between  25°  and  30°  C. 

Upon  agar  and  gelatin  it  grows  with  a  heavy  white  growth,  the 
colonies  of  which  have  a  tendency  to  confluence  and  are  more  mucoid 
in  appearance  than  are  those  of  Bacillus  coli. 

In  hroth,  it  causes  a  general  clouding  and  a  pellicle.  The  cultures 
have  a  slightly  sour  or  cheesy  odor. 

On  potato,  the  growth  is  heavy  and  gas  is  formed. 

On  milk,  there  is  rapid  coagulation  and  acid  formation.  It  is  charac- 
teristic of  this  bacillus  that  it  is  capable  of  producing  a  large  amount  of 
acid,  chiefly  lactic,  and  of  being  able  to  withstand  these  large  amounts 
of  acid  without  being  injured  by  them. 


1  Escherich,  Fort.  d.  Med.,  16,  17,  1885.    2  Wilde,  Cent,  f .  Bakt.,  xx,  1896.    ^  Jackson. 


454  PATHOGENIC  MICROORGANISMS 

The  pathogenicity  of  Bacillus  lactis  aerogenes  for  man  is  slight. 
Its  chief  claims  to  importance  lie  in  its  milk-coagulating  properties 
and  its  almost  constant  presence  in  the  human  intestine.  In  infants,  it 
may  give  rise  to  flatulence  and  it  has  been  occasionally  observed  as  the 
sole  incitant  of  cystitis.  Among  such  cases  rare  instances  have  been 
observed  in  which  it  has  formed  gas  in  the  bladder  (pneumaturia). 
When  this  occurs  the  urine  is  not  ammoniacal  but  remains  acid. 

Different  strains  of  this  bacillus  vary  much  in  their  pathogenicity 
for  animals.  Wilde  claims  that  it  is  more  pathogenic  for  white  mice  and 
gionea-pigs  than  is  the  bacillus  of  Friedlander.  He  speaks  of  it  as  the 
most  virulent  member  of  this  group.  Kraus,  writing  in  Fluegge's 
"Mikroorganismen/'  rates  its  pathogenicity  less  high. 

Closely  related  to  this  bacillus,  as  well  as  to  those  of  the  Friedlander 
group,  is  an  encapsulated  bacillus  isolated  from  a  case  of  broncho- 
pneumonia by  Mallory  and  Wright,^  which  is  strongly  pathogenic  for 
mice,  guinea-pigs,  and  rabbits. 

BACILLI  OP  THE  PROTEUS  GROUP 

,  The  bacilli  of  this  group  have  little  pathological  interest,  but  are  im- 
portant because  of  the  frequency  with  which  they  are  encountered  in 
routine  bacteriological  work.  They  may  confuse  the  inexperienced 
because  of  a  superficial  similarity  to  bacilli  of  the  colon-typhoid  group. 
In  form  they  may  be  short  and  plump  or  long  and  slender,  staining  easily 
with  anilin  dyes  and  decolorizing  with  Gram's  method.  They  are  ac- 
tively motile  and  possess  many  flagella.  Individuals  stain  irregularly, 
often  showing  unstained  areas  near  the  center.  The  so-called  Bacillus 
proteus  vulgaris  described  by  Hauser  ^  m  1885  is  the  type  of  the  group. 

Bacilli  of  this  group  are  widely  distributed,  being  found  in  water, 
soil,  air,  and  wherever  putrefaction  takes  place.  In  fact,  proteus  is  one 
of  the  true  putrefactive  bacteria  possessing  the  power  to  cause  the  cleav- 
age of  proteids  into  their  simplest  radicles. 

Bacillus  proteus  vulgaris  grows  best  at  temperatures  at  or  about 
25°  C.  and  develops  upon  the  simplest  media.  It  is  a  facultative  anae- 
robe and  forms  no  spores.  In  hroth,  it  produces  rapid  clouding  with  a 
pellicle  and  the  formation  of  a  mucoid  sediment.  In  gelatin,  the  colonies 
are  characteristically  irregular,  giving  the  name  to  this  group. 

Gelatin  is  rapidly  liquefied.  Liquefaction,  however,  is  diminished 
or  even  inhibited  under  anaerobic  conditions. 

1  Mallory  and  Wright,  Zeit.  f.  Hyg.,  xx,  1895,   . 
^Hauser,  "Ueber  Faulniss-Bakt.,"  Leipzig,  1885. 


BACILLUS  PROTEUS  455 

On  agar  and  other  solid  media,  as  well  as  upon  gelatin  before  lique- 
faction has  taken  place,  characteristic  colonies  are  produced.  From 
the  central  flat,  grayish-white  colony  nucleus,  numerous  irregular 
streamers  grow  out  over  the  surrounding  media,  giving  the  colony  a 
stellate  appearance.  On  potato,  it  forms  a  dirty,  yellowish  growth. 
In  milkf  there  is  coagulation  and  an  acid  reaction  at  first;  later  the 
casein  is  redissolved  by  proteolysis.  Blood  serum  is  often  liquefied, 
but  not  by  all  races. 

A  great  many  really  dissimilar  bacteria  have  been  described  under 
the  name  of  Proteus.  The  type  of  the  group  is  the  so-called  Proteus 
vulgaris  (Hauser,  1885).  Other  organisms  spoken  of  as  proteus  are 
the  Proteus  mirahilis,  which  differs  in  slower  gelatin  liquefication 
from  vulgaris,  the  Proteus  Zenkeri,  which  does  not  liquefy  gelatin, 
the  Proteus  septicus,  and  the  Bacillus  Zopfi,  a  Gram-positive  organ- 
ism. A  good  many  of  these  were  formerly  classified  as  of  Bacterium 
termo.  Closely  related  is  the  slow  liquefying  organism  known  as 
Bacillus  cloacce,  common  in  sewage. 

There  is  no  group  which  so  urgently  requires  study  as  this,  since 
organisms  belonging  here  are  so  often  found  in  the  human  body  and 
human  excreta.  In  urine  we  have  encountered  a  non-gelatin  liquefy- 
ing Gram-negative  bacillus  belonging  to  this  group  which  has  given 
us  much  trouble  in  identification.  As  far  as  we  can  establish  any 
general  characteristics  for  the  group  at  all,  we  may  say  that  they  are 
Gram-negative,  non-spore-bearing,  motile  bacilli,  which  on  the  surface 
of  gelatin  plates  show  colonies  characterized  by  spreading  streamers, 
most  of  which  liquefy  gelatin,  a  few  of  which,  however,  do  not.  All 
of  them  ferment  dextrose  and  saccharose  with  gas,  but  do  not  attack 
lactose. 

The  pathogenic  powers  of  proteus  are  slight.  Large  doses  injected 
into  animals  m^y  give  rise  to  localized  abscesses.  In  man  proteus  in- 
fections have  been  described  in  the  bladder,  in  most  cases,  however, 
together  with  some  other  microorganism.  The  Urohacillus  lique- 
faciens  septicus  described  by  Krogius  was  a  variety  of  this  group. 
Epidemics  ^  of  meat  poisoning  have  been  attributed  to  the  proteus 
family  by  some  observers.  Thus  Wesenberg^  cultivated  a  proteus 
from  putrid  meat  which  had  caused  acute  gastroenteritis  in  sixty- 
three  individuals.  Similar  epidemics  have  been  reported  by  Silber- 
schmidt,  ^  Pfuhl,  *  and  others. 

1  Schnitzler,  Cent,  f .  Bakt.,  viii,  1890.  2  Wesenberg,  Zeit.  f.  Hyg.,  xxviii,  1898. 

3  Silberschmidt,  Zeit.  f .  Hyg.,  xxx,  1899.    _  *  Pfuhl,  Zeit.  f,  Hyg.,  xxxv,  1900. 


CHAPTER  XXXI 
BACILLUS  TETANI 

Lockjaw  or  tetanus,  though  a  comparatively  infrequent  disease, 
has  been  recognized  as  a  distinct  clinical  entity  for  many  centuries. 
The  infectious  nature  of  the  disease,  however,  was  not  demonstrated 
until  1884,  when  Carlo  ^  and  Rattone  succeeded  in  producing  tetanus  in 
rabbits  by  the  inoculation  of  pus  from  the  cutaneous  lesion  of  a  human 
case.  Nicolaier,^  not  long  after,  succeeded  in  producing  tetanic  symp- 
toms in  mice  and  rabbits  by  inoculating  them  with  soil.  In  connec- 
tion with  the  lesions  produced  at  the  point  of  inoculation,  Nicolaier 
described  a  bacillus  which  may  have  been  Bacillus  tetani,  but  which  he 
was  unable  to  cultivate  in  pure  culture.  Kitasato,^  in  1889,  definitely 
solved  the  etiological  problem  by  obtaining  from  cases  of  tetanus  pure 
cultures  of  bacilli  with  which  he  was  able  again  to  produce  the  disease 
in  animals. 

Kitasato  succeeded  where  others  had  failed  because  of  his  use  of 
anaerobic  methods  and  his  elimination  of  non-spore-bearing  con- 
taminating organisms  by  means  of  heat.  His  method  of  isolation 
was  as  follows:  The  material  containing  tetanus  bacilli  was  smeared 
upon  the  surface  of  agar  slants.  These  were  permitted  to  develop  at 
incubator  temperature  for  twenty-four  to  forty-eight  hours.  At  the  end 
of  this  time  the  cultures  were  subjected  to  a  temperature  of  80^  C.  for 
one  hour.  The  purpose  of  this  was  to  destroy  all  non-sporulating 
bacteria,  as  well  as  aerobic  spore-bearers  which  had  developed  into 
the  vegetative  form.  Agar  plates  were  then  inoculated  from  the  slants 
and  exposed  to  an  atmosphere  from  which  oxygen  had  been  com- 
pletely eliminated  and  hydrogen  substituted.  On  these  plates  colonies 
of  tetanus  bacilli  developed. 

Morphology  and  Staining. — ^The  bacillus  of  tetanus  is  a  slender  bacil- 
lus, 2  to  5  micra  in  length,  and  0.3  to  0.8  in  breadth.  The  vegetative 
forms  which  occur  chiefly  in  young  cultures  are  slightly  motile  and  are 

»  Carlo  e  Rattone,  Giomale  d.  R.  Acad.  d.  Torino,  1884. 
'  Nicolaier,  Inaug,  Diss,,  Gottingen,  1885. 
8  Kitasato,  Deut.  med.  Woch.,  No.  xxxi,  1889. 
456 


BACILLUS  TETANI 


457 


seen  to  possess  ^  numerous  peritrichal  flagella,  when  stained  by  special 
methods.  After  twenty-four  to  fgrty-eight  hours  of  incubation,  the 
length  of  time  depending  somewhat  on  the  nature  of  the  medium  and 
the  degree  of  anaerobiosis,  the  bacilli  develop  spores  which  are  char- 
acteristically located  at  one  end,  giving  the  bacterium  the  diagnostic 
drumstick  appearance. 

As  the  cultures  grow  older  the  spore-bearing  forms  completely  super 


Fig.  96. — ^Bacillus  tetani.    Spore  stain. 

sede  the  vegetative  ones.  Very  old  cultures  contain  spore-bearing  bacilli 
and  spores  only. 

The  tetanus  bacillus  is  easily  stained  by  the  usual  anilin  dyes,  and 
reacts  positively  to  Gram's  stain.  Flagella  staining  is  successful  only 
when  very  young  cultures  are  employed. 

Distribution. — In  nature,  the  tetanus  bacillus  has  been  found  by 
Nicolaier  and  others  to  occur  in  the  superficial  layers  of  the  soil.    The 


1  Vottaler,  Zeit.  f.  Hyg.,  xxvii. 


458 


PATHOGENIC   MICROORGANISMS 


earth  of  cultivated  and  manured  fields  seems  to  harbor  tnis  organism 
with  especial  frequency,  probably  because  of  its  presence  in  the  dejecta 
of  some  of  the  domestic  animals. 

Biological  Characteristics. — The  bacillus  of  tetanus  is  generally  de- 
scribed as  an  obligatory  anaerobe.  While  it  is  unquestionably  true 
that  growth  is  ordinarily  obtained  only  in  the  complete  absence  of 
oxygen,  various  observers,  notably  Ferran  ^  and 
Belfanti,^  have  successfully  habituated  the  bacillus 
to  aerobic  conditions  by  the  gradual  increase  of 
oxygen  in  cultures.  Habituation  to  aerobic  condi- 
tions has  usually  been  accompanied  by  diminution 
or  loss  of  pathogenicity  and  toxin-formation. 
Anaerobic  conditions  may  likewise  be  dispensed 
with  if  tetanus  bacilli  be  grown  in  symbiosis  with 
some  of  the  aerobic  bacteria.  The  addition  to 
culture  media  of  suitable  carbohydrates,  and  of 
fresh  sterile  liver  tissue,  has  also  been  found  to 
render  it  less  exacting  as  to  absolute  anaerobiosis.^ 

Anaerobically  cultivated.  Bacillus  tetani  grows 
readily  upon  meat-infusion  broth,  which  it  clouds 
within  twenty-four  to  thirty-six  hours.  Anaerobic 
broth  cultures  may  be  simply  made  by  covering  the 
surface  of  the  medium  with  a  layer  of  albolin  or 
any  other  oil,  and  removing  the  air  by  boiling. 

Upon  meat-infusion  gelatin  at  20°  to  22°  C.  the 
tetanus  bacillus  grows  readily,  ■  growth  becoming 
visible  during  the  second  or  third  day.  There  is 
slow  fluidification  of  the  gelatin. 

On  agar,  at  37.5°  C,  growth  appears  within  forty- 
eight  hours.  Colonies  on  agar  plates  present  a  rather 
characteristic  appearance,  consisting  of  a  compact 
center  surrounded  by  a  loose  meshwork  of  fine  fila- 
ments, not  unlike  the  medusa-head  appearance  of  subtilis  colonies. 
In  agar  stabs,  fine  radiating  processes  growing  out  in  all  directions 
from  the  central  stab  tend  to  give  the  culture  the  appearance  of  a  fluff 
of  cotton.  Milk  is  a  favorable  culture  medium  and  is  not  coagulated. 
On  potato,  growth  is  delicate  and  hardly  visible. 

1  Ferran,  Cent.  f.  Bakt.,  xxiv,  No.  1. 

2  Belfanti,  Arch,  per  le  sci.  med.,  xvi. 

«  Th.  Smith,  Brown,  and  Walker,  Jour.  Med.  Res,,  N,  S.,  ix,  1906. 


Fig.  97. — Young 
Tetanus  Culture 
IN  Glucose  Agar. 


BACILLUS  TETANI 


459 


The  most  favorable  temperature  for  the  growth  of  this  bacillus  is 
37.5°  C.  Shght  alkalinity  or  neutrality  of  the  culture  media  is  most  ad- 
vantageous, though  moderate  acidity  does  not  altogether  inhibit  growth. 
All  the  media  named  may  be  rendered  more  favorable  still  by  the  ad- 
dition of  one  or  two  per  cent  of  glucose,  maltose,  or  sodium  formate.^ 
In  media  containing  certain  carbohydrates,  tetanus  bacilli  produce  acid. 
In  gelatin  and  agar,  moderate  amounts  of  gas  are 
produced,  consisting  chiefly  of  CO 2,  but  with  the 
admixtures  of  other  volatile  substances  which  give 
rise  to  a  characteristically  unpleasant  odor,  not  unlike 
that  of  putrefying  organic  matter.  This  odor  is  due 
largely  to  H2  S  and  methylmercaptan. 

The  vegetative  forms  of  the  tetanus  bacillus  are 
not  more  resistant  against  heat  or  chemical  agents 
than  the  vegetative  forms  of  other  microorganisms. 
Tetanus  spores,  however,  will  resist  dry  heat  at 
80°  C.  for  about  one  hour,  live  steam  for  about 
five  minutes;  five  per  cent  carbolic  acid  kills  them 
in  twelve  to  fifteen  hours;  one  per  cent  of  bichlo- 
rid  of  mercury  in  two  or  three  hours.  Direct 
sunlight  diminishes  their  virulence  and  eventually 
destroys  them.^  Protected  from  sunlight  and  other 
deleterious  influences,  tetanus  spores  may  remain 
viable  and  virulent  for  many  years.  Henrijean  * 
has  reported  her  success  in  producing  tetanus  with 
bacilli  from  a  splinter  of  wood  infected  eleven 
years  before. 

Pathogenicity. — The  comparative  infrequency  of 
tetanus  infection  is  in  marked  contrast  to  the  wide 
distribution  of  the  bacilli  in  nature.    Introduced  into 
the  animal  body  as  spores,  and  free  from  toxin,  they 
may  often  fail  to  incite  disease,  easily  falling  prey  to 
phagocytosis  and  other  protective  agencies  before  the  vegetative  forms 
develop  and  toxin  is  formed.     The  protective  importance  of  phagocyto- 
sis was  demonstrated  by  Vaillard  and  Rouget,'*  who  introduced  tetanus 
spores  inclosed  in  paper  sacs  into  the  animal  body.      By  the  paper  cap- 

1  Kitasato,  Zeit.  f.  Hyg.,  1891. 

2v.  Eisler  und  Pribram,  in  Levaditi,  "Handbuch,"  etc.,  Jena,  1907. 

*  Henrijean,  Ann.  de  la  soc.  med.  chir.  de  Liege,  1891. 

*  Vaillard  et  Rouget,  Ann.  de  I'inst.  Pasteur,  1892. 

30 


Fig.  98.— Older 
Tetanus  Culture 
IN  Glucose  Agar. 


460  PATHOGENIC  MICROORGANISMS 

sules  the  spores  were  protected  from  the  leucocytes,  not  from  the  body 
fluids.  Nevertheless,  tetanus  developed  in  the  animals.  The  nature  of 
the  wound  and  the  simultaneous  presence  of  other  microorganisms  seem 
to  be  important  factors  in  determining  whether  or  not  the  tetanus  bacilli 
shall  be  enabled  to  proliferate.  Deep,  lacerated  wounds,  in  which  there 
has  been  considerable  tissue  destruction,  and  in  which  chips  of  glass, 
wood  splinters,  or  grains  of  dirt  have  become  embedded,  are  particularly 
favorable  for  the  development  of  these  germs.  The  injuries  of  compound 
fractures  and  of  gunshot  wounds  are  especially  liable  to  supply  these 
conditions,  and  the  presence  in  such  wounds  of  the  common  pus  cocci, 
or  of  other  more  harmless  parasites,  may  aid  materially  in  furnishing  an 
environment  suitable  for  the  growth  of  the  tetanus  bacilli.  Apart  from 
its  occurrence  following  trauma,  tetanus  has  been  not  infrequently  ob- 
served after  childbirth,^  and  isolated  cases  have  been  reported  in  which 
it  has  followed  diphtheria  and  ulcerative  lesions  of  the  throat.^ 

A  definite  period  of  incubation  elapses  between  the  time  of  infection 
with  tetanus  bacilli  and  the  development  of  the  first  symptoms.  In 
man  this  may  last  from  five  to  seven  days  in  acute  cases,  to  from  four 
to  five  weeks  in  the  more  chronic  ones.  Experimental  inoculation  (  f 
guinea-pigs  is  followed  usually  in  from  one  to  three  days  by  rigidity  of 
the  muscles  nearest  the  point  of  infection .  This  spastic  condition  rapidly 
extends  to  other  parts  and  finally  leads  to  death,  which  occurs  within 
four  or  five  days  after  infection. 

Autopsies  upon  human  beings  or  animals  dead  of  tetanus  reveal  few 
and  insignificant  lesions.  The  initial  point  of  infection,  if  at  all  evident, 
is  apt  to  be  small  and  innocent  in  appearance.  Further  than  a  general 
and  moderate  congestion,  the  organs  show  no  pathological  changes. 
BacilH  are  found  sparsely  even  at  the  point  of  infection,  and  have  been 
but  rarely  demonstrated  in  the  blood  or  viscera.  Nicolaier  succeeded 
in  producing  tetanus  with  the  organs  of  infected  animals  in  but  eleven 
out  of  fifty-two  cases.  More  recently,  Tizzoni  ^  and  Creite  ^  have  suc- 
ceeded in  cultivating  tetanus  bacilli  out  of  the  spleen  and  hearths  blood 
of  infected  human  beings. 

The  researches  of  Tarozzi  ^  and  of  Canfora  ^  have  shown  also  that 
spores  may  be  transported  from  the  site  of  inoculation  to  the  liver, 
spleen,  and  other  organs,  and  there  he  dormant  for  as  long  as  fifty-one 
days.    If  injury  of  the  organ  is  experimentally  practised  and  dead  tissue 

1  Baginsky,  Deut.  med.  Woch.,  1893.  ^  Creite,  Cent,  f .  Bakt.,  xxxvii. 

2  Foges,  Wien.  med.  Woch.,  1895.  ^  Tarozzi,  Cent,  f,  Bakt.  Orig.  xxxviii. 
'  Tizzoni,  Ziegler's  Beit.,  vii.  ^  Canfora,  Cent,  f .  Bakt.*  Orig.  xlv. 


BACILLUS   TETANI  461 

6r  blood  clot  produced,  the  spores  may  develop  and  tetanus  ensue. 
These  experiments  may  explain,  cases  of  so-called  cryptogenic  tetanus. 

Tetanus  Toxin. — The  pathogenicity  of  the  tetanus  bacillus  depends 
entirely  upon  the  soluble  toxin  which  it  produces.  This  toxin  is  produced 
in  suitable  media  by  all  strains  of  virulent  tetanus  bacilli,  individual 
strains  showing  less  variation  in  this  respect  than  do  the  separate  strains 
of  diphtheria  bacilli.  While  partial  aerobiosis  does  not  completely  elimi- 
nate toxin  formation,  anaerobic  conditions  are  by  far  more  favorable  for 
its  development. 

The  jnedium  most  frequently  employed  for  the  production  of  tetanus 
toxin  is  neutral  or  slightly  alkaline  beef -infusion  bouillon  containing  five- 
tenths  per  cent  NaCl  and  one  per  cent  pepton.  Glucose,  sodium  formate, 
or  tincture  of  litmus  may  be  added,  but  while  these  substances  increase 
the  speed  of  growth  of  the  bacilli  they  do  not  seem  to  enhance  the  de- 
gree of  toxicity  of  the  cultures.  Glucose  is  said  even  to  be  unfavorable  for 
strong  toxin  development.  It  is  important,  too,  that  the  bouillon  shall 
be  freshly  prepared.^  There  does  not  seem  to  be  any  direct  relationship 
between  the  amount  of  growth  and  the  degree  of  toxicity  of  the  cultures. 
Under  anaerobic  conditions  in  suitable  bouillon  and  grown  at  37.5°  C, 
the  maximum  toxin  content  of  the  cultures  is  reached  in  from  ten  days 
to  two  weeks.    After  this  time  the  toxin  deteriorates  rapidly. 

Tetanus  toxin  has  been  produced  without  resort  to  anaerobic 
methods  by  several  observers,  notably  by  Debrand,^  by  cultivating  the 
bacilli  in  bouillon  in  sjrmbiosis  with  Bacillus  subtilis.  By  this  method, 
Debrand  claims  to  have  produced  toxin  which  was  fully  as  potent  as 
that  produced  by  anaerobic  cultivation. 

The  tetanus  toxin,  in  solution  in  the  bouillon  cultures,  may  be  sepa- 
rated from  the  bacteria  by  filtration  through  Berkefeld  or  Chamberland 
filters.  Since  the  poison  in  such  filtrates  deteriorates  very  rapidly, 
much  more  rapidly  even  than  diphtheria  toxin,  various  methods  have 
been  devised  to  obtain  the  toxin  in  the  solid  state.  The  most  useful  of 
these  is  precipitation  of  the  poison  out  of  solution  by  oversaturation 
with  ammonium  sulphate.^  Very  little  of  the  toxin  is  lost  by  this  method 
and,  thoroughly  dried  and  stocked  in  vacuum  tubes,  together  with  an- 
hyd]X)us  phosphoric  acid,  it  may  be  preserved  indefinitely  without  dete- 
rioration.    The  precipitate  thus  formed  is  easily  soluble  in  water  or 


1  Vaillard  et  Vincent,  Ann.  de  I'inst.  Pasteur,  1891. 

2  Debrand,  Ann.  de  I'inst.  Pasteur,  1890,  1902. 

3  Brieger  und  Cohn,  Zeit.  f .  Hyg.,  xv. 


462  PATHOGENIC  MICROORGANISMS 

salt  solution,  and  therefore  permits  of  the  preparation  of  uniform  solu- 
tions for  purposes  of  standardization. 

Brieger  and  Boer  ^  have  also  succeeded  in  precipitating  the  toxin 
out  of  broth  solution  with  zinc  chloride.  Vaillard  and  Vincent  ^  have 
procured  it  in  the  dry  state  by  evaporation  in  vacuo. 

Brieger  and  Cohn,^  Brieger  and  Boer,^  and  others  have  attempted 
to  isolate  tetanus  poison,  removing  the  proteids  from  the  ammonium  sul- 
phate precipitate  by  various  chemical  methods.  The  purest  preparations 
obtained  have  been  in  the  form  of  fine  yellowish  flakes,  soluble  in  water, 
insoluble  in  alcohol  and  ether.  Solutions  of  this  substance  have  failed 
to  give  the  usual  proteid  reactions. 

The  toxin  when  in  solution  is  extremely  sensitive  to  heat.  Kita- 
sato  ^  states  that  exposure  to  68°  C.  for  five  minutes  destroys  it  com- 
pletely. Dry  toxin  is  more  resistant,^  often  withstanding  temperatures 
of  120°  C.  for  more  than  fifteen  minutes.  Exposure  to  direct  sunlight 
destroys  the  poison  in  fifteen  to  eighteen  hours. ^ 

Interesting  experiments  as  to  the  action  of  eosin  upon  tetanus  toxin 
have  been  carried  out  by  various  observers.  Flexner  and  Noguchi  ^ 
found  that  five  per  cent  eosin  added  to  the  toxin  would  destroy  it 
within  one  hour.  This  action  is  ascribed  to  the  photodynamic  power 
of  the  eosin. 

The  toxin  exerts  an  extremely  low  osmotic  pressure  and  is  easily 
destroyed  by  electric  currents. 

Tetanus  toxin  is  one  of  the  most  pov/erful  poisons  known  to  us. 
Filtrates  of  broth  cultures,  in  quantities  of  0.000,005  c.c,  will  often  prove 
fatal  to  mice  of  ten  grams  weight.  Dry  toxin  obtained  by  ammo- 
nium sulphate  precipitation  ^  is  quantitatively  even  stronger,  values  of 
0.000,001  grams  as  a  lethal  dose  for  a  mouse  of  the  given  weight  not 
being  uncommon.  Brieger  and  Cohn^^  succeeded  in  producing  a  dry 
toxin  capable  of  killing  mice  in  doses  of  0.000,000,05  gram. 

Different  species  of  animals  show  great  variation  in  their  suscepti- 

1  Brieger  und  Boer,  Zeit.  f .  Hyg.,  xxi. 

2  Vaillard  et  Vincent,  Ann.  de  I'inst.  Pasteur,  1891. 
2  Brieger  und  Cohn,  loc.  cit. 

*  Brieger  und  Boer,  Zeit.  f .  Hyg.,  xxi. 
^  Kitasato,  Zeit.  f .  Hyg.,  x. 
^  Morax  et  Marie,  Ann.  de  I'inst.  Pasteur,  1902. 
'  Fermi  und  Pemossi,  Cent,  f .  Bakt.,  xv. 

8  Flexner  and  Noguchi,  "Studies  from  Rockefeller  Inst.,"  v.,  1905. 
^  Brieger  und  Cohn.,  loc.  cit. 
^^  Brieger  und  Cohn.,  Zeit.  f.  Hyg.,  xv. 


BACILLUS  TETANI  463 

bility  to  tetanus  toxin.  Human  beings  and  horses  are  probably  the  most 
susceptible  species  in  proportion  to  their  body  weight.  The  common 
domestic  fowls  are  extremely  resistant.  Calculated  for  grams  of  body 
weight,  the  horse  is  twelve  times  as  susceptible  as  the  mouse,  the  guinea- 
pig  six  times  as  susceptible  as  the  mouse.  The  hen,  on  the  other  hand, 
is  200,000  times  more  resistant  than  the  mouse. 

After  the  inoculation  of  an  animal  with  tetanus  toxin  there  is  always 
a  definite  period  of  incubation  before  the  toxic  spasms  set  in.  This 
period  may  be  shortened  by  increase  of  the  dose,  but  never  entirely 
eliminated.^  When  the  toxin  is  injected  subcutaneously,  spasms  begin 
first  in  the  muscles  nearest  the  point  of  inoculation.  Intravenous 
inoculation,^  on  the  other  hand,  usually  results  in  general  tetanus  of 
all  the  muscles.  The  feeding  of  toxin  does  not  produce  disease,  the 
poison  being  passed  through  the  bowel  unaltered. 

The  harmful  action  of  tetanus  toxin  is  generally  attributed  to  its 
affinity  for  the  central  nervous  system.  Wassermann  and  Takaki  ^ 
show  that  tetanus  toxin  was  fully  neutralized  when  mixed  with  brain 
substance.  Other  organs — liver  and  spleen,  for  instance — showed  no  such 
neutralizing  power.  The  central  origin  of  the  tetanic  contractions  was 
made  very  evident  by  the  work  of  Gumprecht,'^  who  succeeded  in  stop- 
ping the  spasms  in  a  given  region  by  division  of  the  supplying  motor 
nerves. 

The  manner  in  which  the  toxin  reaches  the  central  nervous  system 
has  been  extensively  investigated,  chiefly  by  Meyer  and  Ransom,  and 
Marie  and  Morax.  Meyer  and  Ransom  ^  from  a  series  of  careful  experi- 
ments reached  the  conclusion  that  the  toxin  is  conducted  to  the  nerve 
centers  along  the  paths  of  the  motor  nerves.  Injected  into  the  circu- 
lation,® the  toxin  reaches  simultaneously  all  the  motor  nerve  endings," 
producing  general  tetanus.  In  this  case  too,  therefore,  the  poison  from 
the  blood  can  not  pass  directly  into  the  central  nervous  system,  but 
must  follow  the  route  of  nerve  tracts. 

These  observations  have  been  of  great  practical  value  in  that  they 
pointed  to  the  desirability  of  the  injection  of  tetanus  antitoxin  directly 
into  the  nerves  and  the  central  nervous  system  in  active  cases. 

1  Courmont  et  Doyen,  Arch,  de  phys.,  1893. 

2  Ransom,  Deut.  med.  Woch.,  1893. 

'  Wassermann  und  Takaki,  Berl.  klin.  Woch.,  1898. 
*  Gumprecht,  Pfliiger's  Arch.,  1895. 

^  Meyer  und  Ransom,  Arch,  f .  exp.  Pharm.  u.  Path.,  xlix. 
«  Marie  et  Morax,  Ann.  de  I'inst.  Pasteur,  1902. 


464  PATHOGENIC  MICROORGANISMS 

Tetanolysin. — Tetanus  bouillon  contains,  besides  the  "tetano- 
spasmin*'  described  above  which  produces  the  familiar  symptoms  of  the 
disease,  another  substance  discovered  by  Ehrlich  ^  and  named  by  him 
"tetanolysin."  Tetanolysin  has  the  power  of  causing  hemolysis  of 
the  red  blood  corpuscles  of  various  animals,  and  is  an  entirely  separate 
substance  from  tetanospasmin.  It  may  be  removed  from  toxic  broth 
by  admixture  of  red  blood  cells,  is  more  thermolabile  than  the  tetano- 
spasmin, and  gives  rise  to  an  antihemolysin  when  injected  into  animals. 
For  the  production,  standardization,  and  use  of  tetanus  antitoxin,  see 
p.  220  et  seq. 

Therapeutic  Value  of  Tetanus  Antitoxin. — Until  recently  tetanus 
antitoxin  was  chiefly  useful  as  a  prophylactic,  from  1,000  to  5,000  units 
being  given  by  deeply  subcutaneous  or  intramuscular  injection.  Its 
use  after  the  onset  of  symptoms  had,  however,  been  fraught  with  much 
disappointment,  largely  because  it  was  not  possible  to  influence  the  toxin 
which  had  already  become  united  with  the  substance  of  the  nerve  tis- 
sues. Recently,  however,  more  perfect  methods  of  administration  have 
been  devised  with  which  better  results  have  been  achieved.  The  most 
successful  of  these  seems  to  be  the  method  of  Park  and  Nicoll.^  They 
carry  out  a  spinal  puncture  taking  off  a  moderate  amount  of  spinal  fluid, 
and  then  inject  slowly  by  gravity  from  3,000  to  5,000  units  of  tetanus 
antitoxin  in  a  volume  of  from  3  to  10  c.c.  At  the  same  time  10,000 
units  are  given  intravenously  or  intramuscularly. 

1  Ehrlich,  Berl.  klin.  Woch.,  1898. 

2  Park  and  Nicoll,  Jour,  of  the  A.  M.  A.,  vol.  63,  July,  1914,  p.  235. 


CHAPTER   XXXII 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX,  BACILLUS  OF  MALIGNANT 

EDEMA,  BACILLUS  AEROGENES  CAPSULATUS,  BACILLUS 

BOTULINUS 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX 

{Bacillus  anthracis  symptomatid,  Rauschbrand,  Charbon  symptomatique, 
Sarcophysematos  hovis) 

Symptomatic  anthrax  is  an  infectious  disease  occurring  chiefly 
among  sheep,  cattle,  and  goats.  It  is  spoken  of  as  ''quarter-evil"  or 
"blackleg."  The  disease  has  never  been  observed  in  man.  It  was 
formerly  confused  with  true  anthrax,  because  of  a  superficial  similarity 
between  the  clinical  symptoms  of  the  two  maladies.  Bacteriologically, 
the  two  microorganisms  are  in  entirely  different  classes. 

Symptomatic  anthrax  is  of  wide  distribution  and  infection  is  usually 
through  the  agency  of  the  soil  in  which  the  bacillus  is  present,  in  the 
form  of  spores  which  may  retain  viability  for  several  years. 

Morphology  and  Staining. — The  bacillus  of  symptomatic  anthrax 
is  a  bacillus  with  rounded  ends,  being  about  four  to  six  micra  long,  and 
five-tenths  to  six-tenths  micra  wide.  It  is  usually  seen  singly  and  never 
forms  long  chains.  The  bacillus  in  its  vegetative  form  is  actively  motile 
and  possesses  numerous  flagella  placed  about  its  periphery.  In  artificial 
media  it  forms  spores  which  are  oval,  broader  than  the  rod  itself,  and 
placed  near,  though  never  actually  at,  the  end  of  the  bacillary  body. 
This  gives  the  bacillus  a  racket-shaped  appearance. 

It  is  readily  stained  with  the  usual  anilin  dyes,  but  is  easily  decolor- 
ized by  Gram's  method  of  stainii^g.  However,  von  Hibler  claims  that 
when  verv  carefullv  stained  the  bacillus  can  be  shown  to  be  Gram- 
positive — at  least  when  taken  from  the  animal  body.^ 

Cultivation. — The  bacillus  is  a  strict  anaerobe.  It  was  obtained  in 
pure  culture  first  by  Kitasato.^    Under  anaerobic  conditions  it  is  easily 

^  von  Hibler,  Kolle,  Wassermann,  etc.,  p.  792,  vol.  iv. 
2  Kitasato,  Woch.  f .  Hyg.,  1889. 

465 


466  PATHOGENIC  MICROORGANISMS 

cultivated  upon  the  usual  laboratory  media,  all  of  which  are  more 
favorable  after  the  addition  of  glucose,  glycerin,  or  nutrose.  In  all 
media  there  is  active  gas  formation,  which,  owing  to  an  admixture  of 
butyric  acid,  is  of  a  foul,  sour  odor.  The  bacillus  is  not  very  delicate 
in  its  requirements  of  a  special  reaction  of  media,  growing  equally  well 
on  those  slightly  acid  or  slightly  alkaline. 

On  gelatin  plates,  at  20°  C,  colonies  appear  in  about  twenty-four 
hours,  usually  round  or  oval,  with  a  compact  center  about  which  fine 
radiating  filaments  form  an  opaque  halo.     The  gelatin  is  fluidified. 


V 


\ 


Pig.  99. — Bacillus  of  Symptomatic  Anthrax.     (After  Zettnow.) 

Surface  colonies  upon  agar  plates  are  circular  and  made  up  of  a 
slightly  granular  compact  center,  from  which  a  thinner  peripheral  zone 
emanates,  containing  microscopically  a  tangle  of  fine  threads. 

In  agar  stabs,  at  37.5°  C,  growth  appears  within  eighteen  hours, 
rapidly  spreading  from  the  line  of  stab  as  a  diffuse,  fine  cloud.  Gas 
formation,  especially  near  the  bottom  of  the  tube,  rapidly  leads  to  the 
formation  of  bubbles  and  later  to  extensive  splitting  of  the  medium. 
In  gelatin  stab  cultures  growth  is  similar  to  that  in  agar  stabs,  though 
less  rapid. 

Pathogenicity.— Symptomatic  anthrax  bacilli  are  pathogenic  for 
cattle,  sheep,  and  goats.  By  far  the  largest  number  of  cases,  possibly 
the  only  spontaneous  ones,  appear  among  cattle.  Guinea-p'igs  are  very 
susceptible  to  experimental  inoculation.     Horses  are  very  little  suscep- 


BACILLUS  OF   SYMPTOMATIC  ANTHRAX 


467 


tible.  Dogs,  cats,  rabbits,  and  birds  are  immune.  Man  also  appears 
to  be  absolutely  immune.  Spontaneous  infection  occurs  by  the  en- 
trance of  infected  soil  into  abrasions  or  wounds,  usually  of  the  lower 
extremities.  Infection  depends  to  some  extent  upon  the  relative  de- 
gree of  virulence  of  the  bacillus — a  variable  factor  in  this  species. 
Twelve  to  twenty-four  hours  after  inoculation  there  appears  at  the 
point  of  entrance  a  soft,  puffy  swelling,  which  on 
palpation  is  found  to  emit  an  emphysematous  crack- 
ling. The  emphysema  spreads  rapidly,  often  reaching 
the  abdomen  and  chest  within  a  day.  The  course 
of  the  disease  is  extremely  acute,  the  fever  high, 
the  general  prostration  extreme.  Death  may  result 
within  three  or  four  days  after  inoculation. 

At  autopsy  the  swollen  area  is  found  to  be 
infiltrated  with  a  thick  exudate,  blood-tinged  and 
foamy.  Subcutaneous  tissue  and  muscles  are 
edematous  and  crackle  with  gas.  The  internal 
organs  show  parenchymatous  degeneration  and 
hemorrhagic  areas.  The  bacilli,  immediately  after 
death,  are  found  but  sparsely  distributed  in  the 
blood  and  internal  organs,  but  are  demonstrable  in 
enormous  numbers  in  the  edema  surrounding  the 
central  focus. 

If  carcasses  are  allowed  to  lie  unburied  for  some 
time,  the  bacilli  will  attain  a  general  distribution, 
and  the  entire  body  will  be  found  bloated  with*  gas, 
the  organs  filled  with  bubbles.  Practically  identical 
conditions  are  found  after  experimental  inocula- 
tion. 

Toxins. — According  to  the  investigations  of  Le- 
clainche  and  Vallee,^  the  bacillus  of  symptomatic 
anthrax  produces  a  soluble  toxin.  It  is  not  formed 
to  any  extent  in  ordinary  broth,  but  is  formed  in 
considerable  quantities  in  broth  containing  blood  or  albuminous  ani- 
mal fluids. 

The  best  medium  for  obtaining  toxin,  according  to  the  same  authors, 
is  the  bouillon  of  Martin,^  made  up  of  equal  parts  of  veal  infusion  and  a 


Fig.  100.— Ba- 
cillus OF  Symp- 
tomatic Anthkax. 
Culture  in  glucose 
agar. 


1  Leclainche  et  Vallee,  Ann.  de  I'inst.  Pasteur,  1900. 

2  Martin,  Ann.  de  I'inst.  Pasteur,  1898. 


468  PATHOGENIC   MICROORGANISMS 

pepton  solution  obtained  from  the  macerated  tissues  of  the  stomachs  of 
pigs.  The  toxin  contained  in  filtrates  of  such  cultures  is  quite  resistant 
to  heat,  but  rapidly  deteriorates  if  free  access  of  air  is  allowed. 

Immunity. — Active  immunization  against  the  bacillus  of  symptom- 
atic anthrax  was  first  accomplished  by  Arloing  ^  and  his  collaborators 
by  the  subcutaneous  inoculation  of  cattle  with  tissue-extracts  of  in- 
fected animals.  The  work  of  these  authors  resulted  in  a  practical 
method  of  immunization  which  is  carried  out  as  follows: 

Two  vaccines  are  prepared.  Vaccine  I  consists  of  the  juice  of  in- 
fected meat,  dried  and  heated  to  100°  C.  for  six  hours.  Vaccine  II  is  a 
similar  meat-juice  heated  to  90°  C,  for  the  same  length  of  time.  By 
the  heating,  the  spores  contained  in  the  vaccines  are  attenuated  to 
relatively  different  degrees.  Vaccine  I  in  quantities  of  0.01  to  0.02 
c.c.  is  emulsified  in  sterile  salt  solutions  and  injected  near  the  end  of 
the  tail  of  the  animal  to  be  protected.  A  similar  quantity  of  Vaccine  II 
is  injected  in  the  same  way  fourteen  days  later. 

This  method  has  been  retained  in  principle,  but  largely  modified  in 
detail  by  various  workers.  Kitt  ^  introduced  the  use  of  the  dried  and 
powdered  whole  meat  instead  of  the  meat  juice,  and  made  only  one 
vaccine,  heated  to  94°  C,  for  six  hours.  This  method  has  been  largely 
used  in  this  country.^  Passive  immunization  with  the  serum  *  of 
actively  immunized  sheep  and  goats  has  been  used  in  combination  with 
the  methods  of  active  immunization. 


BACILLUS  OF  MALIGNANT  EDEMA 

(Bacillus  oedernatis  maligni,  Vibrion  septique) 

In  1877,  Pasteur  ^  described  a  bacillus  which  he  had  found  in  guinea- 
pigs  and  rabbits  experimentally  inoculated  with  putrefying  animal 
tissues.  This  bacillus,  which  he  named  "Vibrion  septique,"  he  suc- 
ceeded in  cultivating  only  under  anaerobic  conditions  and  in  an  impure 
state,  and  described  as  its  pathognomonic  characteristics  the  formation 
of  an  extensive  edema  in  and  about  the  point  of  inoculation. 

^  Arloing,  Cornevin,  et  Thomas,  "  Le  Charbon  Sympt.,"  etc.,  Paris,  1887.  Kei. 
from  Grassberger  und  Schattenfroh,  Kraus  und  Levaditi,  "Handbuch,"  etc.,  vol 
i,  pt.  2. 

2  Kitt,  Ref .  from  Grassberger  und  Schattenfroh,  loa  cit. 

»  Ti<»nnrt  of  Bureau  of  Animal  Ind.,  Wash.,  1902. 

*  Arloing,  Leclainche,  et  Vallee,  loc.  cit. 

6  Pasteur,  Bull,  de  I'acad.  de  mM.,  1877,  p.  793. 


BACILLUS   OF  MALIGNANT   EDEMA 


469 


Koch/  who  studied  this  infection  in  connection  with  his  work  upon 
anthrax  in  1881,  called  attention  to  the  fact  that  the  bacillus  described 
by  Pasteur  did  not  produce  a  true  septicemia,  and  suggested  the  term 
"Bacillus  of  malignant  edema,"  which  is  now  in  general  use. 

Gaffky  ^  found  that,  apart  from  its  presence  in  putrid  material,  the 
bacillus  occurred  in  the  upper  layers  of  garden  soil  and  in  dust.  It 
has  since  been  found  to  be  widely  distributed  in  nature  and  in  the 
intestines  of  animals  and  of  man.  Its  wide  diistribution  is  unques- 
tionably due  to  the  great  resistance  of  its  spores. 

Morphology  and  Staining. — The  bacillus  of  malignant  edema  is  a 


^^^J 


Fic3.  101. — Bacillus  of  Malignant  Edema.     (After  Frankel  and  Pfeiffer.) 


long  slender  rod,  not  unlike  the  anthrax  bacillus,  but  decidedly  more 
slender.  Its  average  measurements  are  1  micron  in  thickness  and  3 
to  8  micra  in  length.  It  usually  occurs  as  single  rods,  but  frequently 
appears  in  long  threads  showing  irregular  subdivisions.  Often  no  sub- 
divisions can  be  seen  and  the  threads  appear  as  long,  homogeneous 
filaments.  These  threads  are  less  frequently  seen  in  preparations  from 
solid  media  than  in  those  from  bouillon  or  edema  fluid.  'The  bacilli 
are  motile  and  possess  numerous  laterally  placed  flagella.  Their  motil- 
ity is  never  very  marked  and  is   often  entirely  absent.     The  bacillus 

»  Koch,  Mitt.  a.  d.  kais.  Gesundheitsamt,  i,  1881,  p.  52  et  seq. 
2  Gaffky,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1881. 


470 


pathogejnic  microorganisms 


produces  spores  at  temperatures  above  20°  C,  which  are  oval,  irregularly- 
placed  either  in  the  center  or  slightly  nearer  one  or  the  other  end,  and 
cause  a  bulging  of  the  bacillary  body. 

It  is  readily  stained  by  any  of  the  usual  anilin  dyes.  Stained  by 
Gram's  method  it  is  decolorized. 

Cultivation. — Bacillus  oedematis  maligni  is  strictly  anaerobic. 
Under  anaerobic  conditions  it  develops  readily  upon 
any  of  the  usual  artificial  media.  The  bacillus  is  not 
very  sensitive  to  the  reaction  of  media  and  grows 
more  luxuriantly  in  all  media  to  which  glucose  has 
been  added.  In  all  media  it  forms,  by  the  cleavage  of 
proteids,  putridly  offensive  gases. 

In  gelatin  at  room  temperature,  colonies  develop  in 
about  three  days  as  small  grayish  spherical  growths, 
which  microscopically  show  an  arrangement  in  radial 
filaments.     The  gelatin  is  fluidified. 

In  gelatin  stab  cultures  growth  begins  as  a  white 
column  extending  to  within  a  centimeter  of  the  top 
of  the  medium.  Soon  irregularly  radiating  processes 
de^^elop  laterally  and  gas  bubbles  appear,  breaking  up 
the  medium. 

Stab  cultures  in  agar  show  growth  within  twenty- 
four  to  thirty-six  hours  at  37.5°  C,  appearing  at  first 
as  a  white  line,  but  soon  showing  a  cloud-like  lateral 
extension  along  the  entire  line  of  the  stab.  If  sugar 
is  present  bubbles  appear  throughout  the  medium. 

In  broth  there  is  general  clouding  and  a  granular 
sediment;  no  pellicle  is  formed.  Milk  is  slowly  coag- 
ulated. On  blood  serum  growth  is  very  luxuriant. 
On  potato,  a  medium  used  in  the  earliest  studies  of 
the  bacillus  by  Gaffky,  the  bacillus  grows  readily. 

Isolation    may    be    accomplished    by  any  of    the 

ordinary  anaerobic  .  plating    methods.      The   bacillus 

can  usually  be  obtained  for  subsequent  isolation  by 

injection  of  a  susceptible  animal  with  soil,  especially  that  of  gardens  or 

manured  fields. 

Pathogenicity. — The  bacillus  is  pathogenic  for  mice,  guinea-pigs, 
rabbits,  horses,  dogs,  sheep,  pigs,  some  birds,  and  man.  Cattle  were 
formerly  regarded  as  immune,  an  opinion  which  has  since  been  found  to 
Vie  erroneous. 


Fig.  102.— Ba- 
cillus OF  Ma- 
lignant Edema. 
Culture  in  glu- 
cose agar. 


BACILLUS  AEROGENES  CAPSULATUS  471 

Subcutaneous  inoculation  of  pure  culture  into  a  susceptible  subject 
produces,  within  twenty-four  to  thirty-six  hours,  an  acute  edematous 
inflammation  about  the  point  of  inoculation.  The  edema  extends 
throughout  the  subcuticular  and  deeper  layers,  and  consists  of  thin, 
slightly  bloody  fluid.  Neighboring  lymph  nodes  become  swollen  and 
hemorrhagic.  In  the  mixed  infections  of  accidental  inoculation,  but 
more  rapidly  in  experimental  inoculations  with  pure  cultures,  gas  is 
formed  and  consequent  subcutaneous  emphysema.  Together  with  this 
there  are  symptoms  of  general  toxemia.  In  the  smaller  test  animals  this 
disease  is  usually  fatal.  At  autopsy  the  bacilli  are  found  in  the  edema 
fluid  about  the  local  lesion.  At  autopsies  done  soon  after  death,  the 
organisms  are  not  found  in  the  blood  or  internal  organs.  Later  they 
may  be  generally  distributed  throughout  the  body.  In  mice  only  may 
the  bacilli  enter  the  blood  stream  before  death.  The  internal  organs 
of  animals  dead  of  this  infection  usually  show  parenchymatous  degen- 
eration and  occasionally  hemorrhages. 

Malignant  edema  is  not  a  frequent  disease.  It  has  been  occasionally 
observed  in  horses,  in  cattle,  and  in  sheep.  In  man  the  infection  usually 
appears  after  traumatism  or  secondarily  after  compound  fractures  or 
upon  the  site  of  suppurating  wounds.  Isolated  cases  have  been  de- 
scribed as  arising  after  hypodermic  injections.  One  case  has  been 
reported  as  arising  in  the  uterus  after  instrumental  abortion. 

Immunity. — Recovery  from  an  infection  with  the  bacillus  of  malig- 
nant edema  produces  immunity  against  subsequent  infections.^  The 
bacillus  in  fluid  media  produces  small  amounts  of  a  soluble  toxin  which 
in  bacteria-free  filtrates  is  capable  of  killing  guinea-pigs.  Relatively 
large  quantities  of  filtrate  must  be  employed.  Roux  and  Chambcrland,^ 
the  first  to  work  upon  these  toxins,  were  able,  by  means  of  them,  to 
immunize  guinea-pigs.  Similar  immunity  could  be  produced  by  treat- 
ment with  the  toxic,  filtered  sera  of  animals  dead  of  the  disease.^ 

BACILLUS  AEROGENES  CAPSULATUS 

The  bacillus  finds  wide  distribution  in  nature,  being  found  in  soil, 
dust,  and  brackish  water,  and  in  the  normal  intestinal  tracts  of  human 
beings  and  mammals.^ 

^  Arlaing  et  Chxiuveau,  Ann.  de  med,  vet.,  1884. 

2  Rouxet  Chamberland,  Ann.  de  I'inst.  Pasteur,  1887.     ^Sanfelice,  Zeit.  f.  Hyg.,  xiv. 

*  Although  B.  aerogenes  capsulatus  and  B.  phlegmonis  emphysematosa  are  sep- 
arately described  in  many  books,  notably  Migula's  ''System  d.  Bakteriologie,"  the 
microorganisms  have  been  shown  beyond  question  to  be  identical  and  are  acknowl- 
edged to  be  so  by  Frankel  himself. 


472  PATHOGENIC  MICROORGANISMS 

There  has  been  endless  confusion  in  the  classification  of  the  so- 
called  aerogenes  capsulatus  organisms,  a  group  which  has  been  known 
in  different  countries  by  a  large  variety  of  names.  Its  first  description 
is  probably  that  by  Welch  and  Nuttall  ^  in  1892,  who  gave  it  the  name 
B.  aerogenes  capsulatus,  by  which  name,  or  briefly  Welch  bacillus,  it  is 
most  commonly  designated  by  American  bacteriologists.  Similar  or 
identical  organisms  are  tfie  B.  phlegmonis  emphysematosoe  of  Fraenkel, 
the  B.  enteriditis  sporogenes  of  Klein,  the  B.  perf ring  ens  of  Veillon  and 
Zuber,  and  the  Granulobacillus  saccharohutyricus  liquefaciens  immohilis 
of  Schattenfroh  and  Grassberger,  and  a  number  of  others  less  prominent 
in  the  literature. 

The  task  of  classifying  these  various  organisms  is  one  of  great 
technical  difficulty,  and,  as  in  so  many  other  groups  of  bacteria  orig- 
inally described  as  a  single  species,  we  are  now  learning  that  instead 
of  complete  identification  of  individual  isolations,  one  with  another, 
we  can  merely  draw  a  circle  about  a  certain  group,  recognizing  close 
relationship  morphologically,  culturally,  and  in  relation  to  infections  of 
man  and  animals.  After  a  very  careful  study  of  many  different  strains, 
Simmonds  comes  to  the  conclusion  that  the  Welch  bacillus,  so-called, 
is  roughly  identical  with  those  mentioned,  including  also  the  bacilli  once 
associated  by  Achalme  with  rheumatic  infections.  The  term  B.  Welchii, 
he  concludes,  does  not  represent  a  fixed  species  but  a  closely  related  group 
of  bacteria  not  yet  fully  classified  even  by  his  own  extensive  studies. 

It  is  often  hard  to  determine  in  the  case  of  organisms  of  such  varied 
activities  exactly  what  can  be  regarded  as  a  cardinal  characteristic 
important  for  classifying  purposes.  The  organisms  treated  of  in  this 
group  are  all  large  Gram-positive,  non-motile  bacilli,  with  rounded 
ends,  rarely  occurring  in  chains,  and  anaerobic.  Spore  formation  is  in- 
constant and  occurs  only  in  an  alkaline  medium.  When  fermentable 
sugars,  and  consequently  free  acid,  are  present,  no  spore  formation 
takes  place.  Milk  is  fermented  with  the  formation  of  butyric  acid. 
The  intravenous  injection  of  animals,  especially  rabbits,  usually  pro- 
duces death  with  an  enormous  swelling  of  the  body  by  the  formation 
of  gas,  which  burns  with  a  pale  blue  flame.  Capsules  may  be  seen 
when  the  smears  are  made  directly  from  animal  tissues,  but  are  almost 
universally  missed  in  organisms  taken  from  culture. 

As  to  minor  cultural  characteristics,  so  many  variations  are  ob- 
served that  it  is  hardly  worth  while  to  summarize  them. 

*  See  Simmonds,  Monographs  of  the  Rockefeller  Institute,  No.  5,  Sept.  27,  1915. 


BACILLUS  AEROGENES  CAPSULATUS  473 

Pathogenicity. — ^It  is  extremely  interesting  that,  though  a  path- 
ogenic anaerobe,  no  one  has  so  far  been  able  to  show  satisfactorily  the 
production  of  a  true  exotoxin,  though  such  a  claim  has  been  made  sev- 
eral times.  Poisonous  products  that  have  been  obtained  from  cultures 
have  not  conformed  with  the  characteristics  of  true  exotoxins  as  we 
recognize  them  now.  Moreover,  the  reaction  of  toxin-containing  fil- 
trates, as  Simmonds  points  out,  has  been  almost  always  acid,  and  Mc- 
Campbell  and  others  have  suggested  that  the  toxicity  of  such  cultures 
is  due  to  the  presence  of  butyric  acid.  It  is  interesting  in  this  connec- 
tion also  that  Herter,  who  gave  a  great  deal  of  attention  to  the  presence 
of  these  organisms  in  the  bowel  and  attributed  to  them  etiological  rela- 
tionship with  many  intestinal  disorders,  believed  that  diarrhea  following 
intestinal  putrefaction  was  due  to  a  very  large  extent  to  the  irritation 
produced  by  ammonium  butyrate,  the  Welch  bacilli  being-  responsible 
for  the  appearance  of  the  butyric  acid.  ^  '^ 

The  search  for  endotoxins,  too,  seems  to  have  been  unsuccessful, 
and  hemolysins  have  been  irregularly  present  in  cultures  studied  by 
various  investigators. 

Great  differences  in  virulence  have  been  observed  in  the  study  of 
various  strains.  A  good  many  of  the  strains  seem  to  have  great  patho- 
genicity for  the  ordinary  laboratory  animals,  especially  for  guinea-pigs 
and  rabbits.  Yet  certain  non-sporulating  forms  have  shown  little  or 
no  virulence.  However,  variation  in  virulence  seems  to  fluctuate  con- 
siderably according  to  the  culture  medium  and  the  symbiotic  conditions 
under  which  the  organisms  have  been  cultivated.  In  man,  fortunately, 
there  seems  to  be  a  relatively  powerful  resistance  against  Welch  bacillus 
infections.  In  fact,  many  investigators  have  believed  that  the  Welch 
bacillus  is  primarily  a  saprophyte  and  requires  the  presence  of  dead 
tissue  or  physiological  injury  before  it  can  penetrate  and  thrive  in 
the  human  body.  It  has  been  observed  in  gas  cysts  of  the  brain,  in 
"gangrenous"  pneumonia,  in  appendix  abscesses,  in  puerperal  sepsis 
after  abortion,  in  general  septicemia,  and  in  many  other  conditions. 
However,  in  many  of  these  cases  it  may  be  quite  reasonable  to  question 
the  primary  character  of  the  "gas  bacillus"  infection,  and  in  others 
again,  the  Welch  bacillus  may  be  regarded  as  an  ante-mortem  invasion, 
a  supposition  strengthened  by  the  study  of  terminal  infections  made  by 
Flexner,  in  which  such  a  possibility  is  plainly  demonstrated.  Thus, 
while  the  Welch  bacillus  may  unquestionably  invade  the  human  body 
and  do  much  injury,  and  even  be  regarded  in  many  cases  as  probably 
the  direct  cause  of  death,  its  invasion  in  many  of  these  cases  must  be 


474  PATHOGENIC  MICROORGANISMS 

regarded  as  not  a  primary  invasion,  but  as  one  associated  with  and 
made  possible  by  either  traumatic  injury  or  the  co-operation  of  other 
invasive  germs  such  as  streptococci,  staphylococci  and  others.  There 
may,  of  course,  in  addition  to  these  be  rkre  instances  in  which  the 
Welch  bacillus,  owing  to  excessive  virulence  of  the  strain  or  relatively 
low  resistance  of  the  infected  individual,  becomes  the  primary  invader. 
It  is  easy  to  understand  under  these  conditions  why  severe  trauma 
associated  with  the  carrying  of  soil  would  form  ideal  conditions  for  the 
production  of  Welch  bacillus  infections. 

The  most  interesting  chapter  in  the  pathogenicity  of  the  Welch 
bacillus  is  its  relation  to  intestinal  disease.  Simmonds  confirms  by  his 
own  work  the  work  of  many  others  that  the  Welch  bacillus  may  be 
regarded  as  a  normal  inhabitant  of  the  intestines  of  man.  In  19  babies 
under  one  year  of  age  he  found  Welch  bacilli  in  the  stools  of  8.  He 
points  out  that  it  has  been  found  even  in  nursing  infants  by  a  number 
of  investigators.  ■ 

Herter  ^  has  associated  the  presence  of  the  Welch  bacillus  in  the 
bowel  with  pernicious  anaemia.  However,  no  such  relationship  can  at 
present  be  assumed  on  the  basis  of  available  work. 

Isolation. — The  bacillus  may,  of  course,  be  isolated  by  anaerobic 
plating  methods.  It  is  best  isolated,  however,  from  mixed  cultures  by 
animal  inoculation.  If,  for  instance,  it  is  desired  to  obtain  it  from  a 
mixed  culture  or  from  feces,  a  suspension  of  about  1  c.c.  of  the  sus- 
pected material  is  made  in  5  c.c.  of  sterile. salt  solution.  This  is  thor- 
oughly emulsified  and  filtered  through  a  sterile  paper.  One  to  two  c.c. 
of  this  suspension  is  then  injected  into  the  ear  vein  of  a  rabbit.  After 
four  or  five  minutes  the  rabbit  is  killed.  It  is  then  placed  in  the  incu- 
bator for  five  to  eight  hours.  At  the  end  of  this  time,  the  animal  is 
usually  found  tensely  distended  with  gas.  At  autopsy,  gas  bubbles 
will  be  found  distributed  throughout  th-e  organs,  most  characteristically 
in  the  liver,  where  isolated  bubbles  are  found  covering  the  surface. 
From  these  bubbles  cultures  or  smears  may  be  taken  for  identification. 
Identification  is  easily  made  from  its  morphology,  its  capsule,  lack  of 
motility,  and  gas  formation. 

The  interest  of  bacteriologists  in  the  Welch  bacillus  has  lately 
been  attracted  particularly  by  the  frequent  occurrence  of  the  co-called 
gas  bacillus  infections  in  soldiers  in  the  trenches.  Apparently,  these 
infections  are  due  to  the  fact  that  dirty  clothing  and  dirt  from  the  skin 

1  Herter,  "  Bacterial  Infection  of  the  Intestinal  Tract,"  New  York,  Macmillan,  1907. 


BACILLUS  BOTULINUS  475 

are  carried  into  the  wounds  with  the  missiles,  and  the  degree  of  trauma 
furnishes  the  conditions  under  which  the  bacillus  can  grow. 

These  infections  are  characterized  by  a  very  marked  destruction  of 
muscle  fibers,  histological  examination  showing  a  complete  disinte- 
gration of  the  cells.  It  is  probable  that  a  great  deal  of  acid,  especially 
butyric  acid,  is  formed  from  the  glycogen  of  the  muscle  by  the  gas 
bacillus,  and  that  this  contributes  to  the  intoxication  of  the  patient. 

Stewart  and  West  have  studied  such  infections  in  guinea-pigs 
in  our  laboratory  and  have  noticed  great  frequency  of  gastric  ulcer 
formation  in  guinea-pigs  succumbing  from  the  bacillus.  Since  they 
could  produce  similar  ulcers  by  intravenous  injections  of  acetic  acid, 
they  concluded  that  much  of  the  systemic  injury  caused  by  the 
bacillus  may  be  associated  with  acid  production. 

Weinberg  and  Sacquep^e  have  recently  claimed  a  true  toxin  in  cul- 
tures isolated  from  wounded  soldiers  and  have  produced  an  intoxication 
against  this  poison.  No  such  true  toxin  has  been  found  in  the  cultures 
studied  in  our  laboratory  and  this,  together  with  the  experiences  of 
many  other  workers,  inclines  us  to  believe  that  the  organism  studied 
by  Weinberg  and  Sacquepee  is  distinct  from  the  bacteria  ordinarily 
classified  with  the  Welch  bacillus. 


BACILLUS  BOTULINUS 

Meat  poisoning  was  formerly  regarded  as  universally  dependent  upon 
putrefactive  changes  taking  place  in  infected  meat,  resulting  in  the 
production  of  ptomains  or  other  harmful  products  of  bacterial  putre- 
faction. It  was  not  until  1888  that  certain  of  these  cases  were  definitely 
recognized  as  true  bacterial  infections,  in  which  the  preformed  poison 
probably  aided  only  in  establishing  the  infection.  Gartner,  in  that 
year,  discovered  the  Bacillus  enteritidis,  a  microorganism  belonging  to 
the  group  of  the  paracolon  bacilli,  and  demonstrated  its  presence  both 
in  the  infecting  meat  and  in  the  intestinal  tracts  of  patients.  The  char- 
acteristics of  this  type  of  meat  poisoning  have  been  discussed  more 
particularly  in  the  section  describing  the  bacillus  of  Gartner  and  its 
allied  forms. 

There  is  another  type  of  meat  poisoning,  however,  which  is  not  only 
much  more  severe  (ending  fatally  in  almost  25  per  cent  of  the  cases),  but 
is  characterized  by  a  clinical  picture  more  significant  of  a  profound 
systemic  toxemia  than  of  a  mere  gastroenteric  irritation.  The  etio- 
logical factor  underlying  this  type  of  infection  was  first  demonstrated 
31 


476  PATHOGENIC  MICROORGANISMS 

by  Van  Ermengem/  in  1896,  and  named  Bacillus  botulinus.  Van 
Ermengem  isolated  the  bacillus  from  a  ham,  the  ingestion  of  which 
had  caused  disease  in  a  large  number  of  persons.  Of  the  thirty-four 
individuals  who  had  eaten  of  it,  all  were  attacked,  about  ten  of  them 
very  severely.  Van  Ermengem  found  the  bacilli  in  large  numbers  lying 
between  the  muscle  fibers  in  the  ham,  and  was  able  to  cultivate  the  same 
microorganism  from  the  stomach  and  spleen  of  one  of  those  who  died 
of  the  infection.  The  results  of  Van  Ermengem  have  been  confirmed 
by  Romer,^  and  others. 

Morphology  and  Staining. — Bacillus  botuHnus  is  a  large,  straight  rod 
with  rounded  ends,  4  to  6  micra  in  length  by  0.9  to  1.2  micra  in  thickness. 
The  bacilli  are  either  single  or  grouped  in  very  short  chains.  Involu- 
tion forms  are  numerous  on  artificial  media.  The  bacillus  is  slightly 
motile  and  possesses  from  four  to  eight  undulated  flagella,  peripherally 
arranged.  Spores  are  formed  in  suitable  media,  most  regularly  in 
glucose-gelatin  of  a  distinctly  alkaline  titer.  The  spores  are  oval  and 
usually  situated  near  the  end  of  the  bacillus,  rarely  in  its  center.  Spores 
are  formed  most  abundantly  when  cultivation  is  carried  out  at  20° 
to  25°  C,  and  are  usually  absent  when  higher  temperatures  are  em- 
ployed. 

The  bacillus  is  easily  stained  by  the  usual  aqueous  aniline  dyes,  and 
retains  the  anilin-gentian-violet  when  stained  by  Gram.  It  is  necessary, 
however,  in  carrying  out  Gram's  stain  to  decolorize  carefully  with  alco- 
hol since  overdecolorization  is  easily  accomplished,  leaving  the  result 
doubtful. 

Cultivation. — The  bacillus  is  a  strict  anaerobe.  In  anaerobic  en- 
vironment it  is  easily  cultivated  on  the  usual  meat-infusion  media.  It 
grows  most  readily  at  temperatures  about  25°  C,  less  luxuriantly  at 
temperatures  of  35°  C.  and  over. 

The  bacillus  is  delicately  susceptible  to  the  reaction  of  media, 
growing  only  in  those  which  are  neutral  or  moderately  alkaline. 

In  deep  stab  cultures  in  one  per  cent  glucose  agar,  growth  is  at  first 
noticed  as  a  thin,  white  column,  not  reaching  to  the  surface  of  the 
medium.  Soon  the  medium  is  cracked  and  split  by  the  abundant 
formation  of  gas.  On  agar  plates,  the  colonies  are  yellowish,  opalescent, 
and  round,  and  show  a  finely  fringed  periphery. 

On  gelatin,  at  20°  to  25°  C.,  growth  is  rapid  and  abundant,  and 

1  Van  Ermengem,  Cent,  f .  Bakt.,  xix,  1896;  Zeit.  f.  Hyg.,  xxvi,  1897. 

2  Romer,  Cent.  f.  Bakt.,  xxvii,  1900. 


BACILLUS  BOTULINUS  477 

differs  little  from  that  oh  agar,  except  that,  besides  the  formation  of 
gas,  there  is  energetic  fluidification  of  the  medium.  On  glucose-gelatin 
plates,  Van  Ermengem  describes  the  colonies  as  round,  yellowish, 
transparent,  and  composed  of  coarse  granules  which,  along  the  periphery 
in  the  zone  of  fluidification,  show  constant  motion.  The  appearance  of 
the  surface  colonies  on  glucose-gelatin  plates  is  regarded  by  the  discov- 
erer as  diagnostically  characteristic. 

In  glucose  hroth  there  is  general  clouding  and  large  quantities  of 
gas  are  formed.  At  35°  C.  and  over,  the  gas  formation  ceases  after  four 
or  five  days,  the  broth  becoming  clear  with  a  yellowish-white  flocculent 
sediment.    At  lower  temperatures  this  does  not  occur. 

Milk  is  not  coagulated  and  disaccharids  and  polysaccharids  are  not 
fermented 

The  gas  formed  in  cultures  consists  chiefly  of  hydrogen  and  methane. 
All  cultures  have  a  sour  odor,  like  butyric  acid,  but  this  is  not  so  offensive 
as  that  of  some  of  the  other  anaerobic  organisms. 

The  bacillus  lives  longest  in  gelatin  cultures,  but  even  upon  these, 
transplantations  sHould  be  done  every  four  to  six  weeks,  since  the 
spores  of  this  bacillus  show  less  viability  and  resistance  than  do  those  of 
most  spore-formers. 

Pathogenicity. — Botulism,  or  allantiasis,  as  noticed  in  human  beings, 
is,  as  far  as  we  know,  always  due  to  ingestion  of  infected  meat,  usually 
of  ham,  canned  meats,  or  sausages  (botulus  =  sausage).  Symptoms 
appear  only  after  a  definite  period  of  incubation  which  varies  from 
twenty-four  to  forty-eight  hours.  The  first  definite  symptoms  are 
chilliness,  trembling,  and  giddiness.  These  manifestations  are  soon 
followed  by  headache,  occasionally  by  vomiting.  In  contradistinction' 
to  the  meat  poisonings  caused  by  other  microorganisms,  those,  due  to 
Bacillus  botulinus  may  show  few  or  no  symptoms  directly  referable  to 
the  intestinal  tract.  The  chief  diagnostic  characteristics  of  the  disease 
are  a  group  of  symptoms  referable  to  toxic  interference  with  the  cranial 
nerves.  Loss  of  accommodation,  dilated  pupils,  ptosis,  aphonia,  and 
dysphagia  may  occur.  Fever  is  usually  absent.  Consciousness  is  rarely 
lost.  The  characteristic  symptoms  may  be  produced  in  various  animals 
by  injection  of  living  cultures  or  culture  filtrates,  i.e.,  toxins.  The  most 
susceptible  animals  are  guinea-pigs.  These  may  be  killed  by  the  injec- 
tion of  minute  quantities  of  bouillon  cultures  or  of  toxin.  Preceding 
death,  which  occurs  within  twenty-four  to  thirty-six  hours,  there  m.ay 
be  general  motor  paralysis,  dyspnea,  and  hypersecretion  of  mucus  from 
nose  and  mouth.    Guinea-pigs  may  be  infected  per  os  as  well  as  by 


478  *"  PATHOGENIC  MICROORGANISMS 

hypodermic  injections.  Cats,  mice,  and  monkeys  are  highly  susceptible; 
rabbits  are  less  so,  but  still  favorable  subjects  for  experimental  studies. 
Birds,  especially  pigeons,  are  highly  resistant,  but  react  typically  to 
large  doses.  Autopsies  upon  man  or  animals  dead  of  botulism  show 
general  hyperemia  of  the  organs  with  much  parenchymatous  degenera- 
tion and  many  minute  hemorrhages. 

The  bacilli  have  been  found  in  the  spleen  after  death,i  but  Van 
Ermengem  does  not  believe  that  they  are  generally  distributed  during 
the  course  of  the  disease.  It  is  believed  by  most  of  those  who  have 
studied  this  disease  that  poisoning  in  the  human  subject  is  due  to  the 
toxins  preformed  and  ingested  within  the  infected  meat  by  this  bacillus. 
Experiments  have  shown  that  little  or  no  poison  is  produced  by  the 
bacilli  after  gaining  entrance  to  the  himian  or  animal  body. 

The  Toxin  of  B.  botulinus — Bacillus  botulinus  produces  disease 
chiefly  by  means  of  a  strong  soluble  toxin  secreted  by  it,  and  absorbed 
by  the  infected  subject.  This  toxin  is  active  in  animals  and  presumably 
in  man,  not  only  when  injected  subcutaneously,  but  also  when  intro- 
duced through  the  gastrointestinal  canal.  The  poison  has  been  par- 
ticularly studied  by  Brieger  and  his  collaborators.  It  is  obtained  in 
filtrates  of  alkaline  bouillon  cultures.  It  has  been  precipitated  out  of 
the  filtrate  by  Brieger  and  Kempner  ^  by  means  of  a  three  per  cent  zinc 
chlorid  solution  (2  volumes  of  3  per  cent  ZnCl2).  The  toxin  thus  ob- 
tained was  sufficiently  powerful  to  kill  a  250-gram  guinea-pig  in  fifty 
hours. 

Specific  action  of  the  toxin  for  the  nerve-cells  of  the  spinal  ganglia 
has  been  shown  by  Marinesco.^  A  specific  antitoxin  has  been  produced 
by  Kempner  and  Pollack.^ 

1  Stricht,  Quoted  from  Van  Ermengem,  in  KoUe  und  Wassermann. 

2  Brieger  und  Kempner,  Deut.  med.  Woch.,  xxxiii,  1897. 

3  Marinesco,  Compt.  rend,  de  I'acad.  des  sci.,  Nov.,  1896. 
*  Kempner  und  Pollack,  Deut.  med.  Woch.,  xxxii,  1897. 


CHAPTER   XXXIII 
THE  TUBERCLE  BACILLUS 

In  view  of  the  clinical  manifestations  of  tuberculosis,  it  is  not  sur- 
prising that  the  infectious  nature  of  the  disease  has  been  suspected  for 
many  centuries.  Even  Fracastor  had  remarkably  modern  ideas  concern- 
ing its  transmission.  Inoculation  by  means  of  tuberculous  material  was 
first  successfully  accomplished  by  Klencke,  in  1843,  and,  more  elabo- 
rately, by  Villemin,^  in  1865.  It  was  not  until  1882,  however,  that 
Koch  2  succeeded  in  isolating  and  cultivating  the  tubercle  bacillus. 
Baumgarten  ^  had  previously  seen  the  bacillus  in  tissue  sections,  but  his 
researches  were  limited  to  purely  moi-phological  observations.  Koch, 
in  addition  to  demonstrating  the  bacillus  in  tuberculous  tissues  from 
various  sources,  produced  characteristic  lesions  in  guinea-pigs  and  other 
animals  by  infecting  them  with  pure  cultures,  and  established  beyond 
doubt  the  etiological  relationship  of  the  bacillus  to  the  disease. 

Morphology. — ^Tubercle  bacilli  appear  as  slender  rods,  2  to  4  micra 
in  length,  0.2  to  0.5  micra  in  width.  Their  ends  are  usually  rounded. 
The  rods  may  be  straight  or  slightly  curved;  their  diameters  may  be 
uniform  throughout;  more  often,  however,  they  appear  beaded  and 
irregularly  stained.  The  beaded  appearance  is  due  to  different  causes. 
Unstained  spaces  may  occur  along  the  body  of  the  bacillus,  especially 
in  old  cultures.  These  are  generally  regarded  as  vacuoles.  The  bodies 
of  the  bacilli,  on  the  other  hand,  may  bulge  slightly  here  and  there,  often 
in  three  or  four  places,  showing  oval  or  rounded  knobs  which  stain  with 
great  depth  and  are  very  resistant  to  decolorization.  These  thickenings 
were  formerly  regarded  as  spores,  but  in  view  of  the  fact  that  the  bacilli 
are  not  more  resistant  against  heat  and  disinfectants  than  other  vegeta- 
tive forms,  this  interpretation  is  probably  incorrect.  The  bacilli  are  said 
to  possess  a  cell  membrane  which  confers  upon  them  their  resistance 
against  drying  and  entrance  of  stains.  This  membrane  gives  a  cellulose 
reaction  and  is  believed  to  contain  most  of  the  waxy  substances  which 
can  be  extracted  from  the  cultures. 

1  Villemin,  Gaz.  hebdom.,  1865. 

2  Koch,  Berl.  klin.  Woch.,  1882;  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 
^Baumgarten,  Virchow's  Arch.,  Ixxxii. 

479 


480 


PATHOGENIC   MICROORGANISMS 


Various  observers,  notably  Nocard  and  Roux/  Mafucci,^  and  Klein,^ 
have  demonstrated  branched  forms  of  the  tubercle  bacillus.  These  ob- 
servations, variously  extended  and  confirmed,  make  it  probable  that 
Bacillus  tuberculosis  is  not  a  member  of  the  family  of  schizomycetes, 


0 

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It     '  -<  i 

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y 

f 

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Fig.  103. — ^Tubercle  Bacilli  in  Sputum. 


but  belongs  rather  to  the  higher  bacteria,  closely  related  to  the  actino- 
myces. 

Staining. — Tubercle  bacilli  do  not  stain  easily  with  the  ordinary 
anilin  dyes;  to  these  they  are  made  permeable  only  by  long  exposure 
or  by  heating  of  the  staining  solution.  Once  stained,  however,  the  dye 
is  tenaciously  retained  in  spite  of  treatment  with  alcohol  and  strong 

1  Nocard  et  Roux,  Ann.  de  I'inst.  Pasteur,  1887.  ^  Mafucci,  Zeit.  f.  Hyg.,  ii. 

8  KUin,  Cent,  f .  Bakt.,  1890. 


THE  TUBERCLE  BACILLUS  481 

acids.  For  this  reason,  this  bacillus,  together  with  some  other  bacteria 
to  be  mentioned  later,  is  spoken  of  as  ''  acid-fast."  The  acid-fast  nature 
of  the  bacillus  seems  to  depend  upon  the  fatty  substances  contained  in 
it,^  and  has  furnished  the  basis  for  differential  staining  methods.  All 
the  staining  methods  devised  for  the  recognition  of  the  tubercle  bacillus 
thus  depend  upon  the  use  of  an  intensely  penetrating  staining  solution, 
followed  by  vigorous  decolorization  which  deprives  all  but  the  acid-fast 
group  of  their  color.  Counterstains  of  any  of  the  weaker  dyes  may 
then  be  used  to  stain  the  decolorized  elements.  One  of  the  first  of  the 
staining  solutions  to  be  of  practical  use  was  the  anilin-water-gentian- 
violet  solution  of  Ehrlich  ^  (11  c.c.  saturated  alcoholic  gentian- violet 
to  89  c.c.  5  per  cent  anilin  water).  This  dye,  although  of  sufficient 
penetrating  power,  has  the  disadvantage  of  deteriorating  rapidly  and 
has  in  practice  been  almost  entirely  displaced  by  ZiehFs  ^  carbol-fuchsin 
solution.  (Fuchsin  1  gm.  in  10  c.c.  alcohol  absolute,  added  to  90  c.c. 
5  per  cent  carbolic.)  This  staining  solution  is  the  one  now  in 
general  use  and  is  employed  as  follows:  Thin  smears,  on  slides 
or  cover-slips,  are  covered  with  the  dye  and  gently  heated.  In 
the  case  of  cover-glasses,  these  may  be  floated,  face  downward, 
in  staining  dishes  filled  with  the  dye.  The  dye  is  allowed  to  act 
for  about  three  miimtes,  steaming  but  not  allowed  to  boil.  At  the 
end  of  this  time  the  preparation  is  washed  either  with  5  per  cent 
nitric  acid,  5  to  20  per  cent  sulphuric  acid,  or  1  per  cent  hydro- 
chloric acid,  until  most  of  the  red  color  has  disappeared  (a  few 
seconds),  and  the  preparation  appears  pale  pink.  This  results  in 
decolorization  of  all  microorganisms  with  the  exception  of  members 
of  the  acid-fast  group.  Thorough  washing  in  80  to  95  per  cent  alcohol 
is  now  employed  to  complete  the  decolorization.  The  preparation 
is  then  rinsed  in  water  and  counterstained  with  1  per  cent  aqueous 
methylene-blue. 

Tubercle-bacillus  staining  has  been  further  simplified  by  Gabbett,'' 
who  combines  decolorization  and  counterstaining.  In  this  method 
preparations  are  stained  with  Ziehl's  carbol-fuchsin  as  in  the  preceding; 
they  are  then  rinsed  in  water  and  covered  with  a  solution  containing 
methylene-blue  1  gram,  concentrated  sulphuric  acid  25  grams,  and 
distilled  water  100  c.c.      This  is  allowed  to  act  for  from  two  to  four 

1  Bienstock,  Fort.  d.  Med.,  1886;   Weyl,  Deut.  med.  Woch.,  1891. 

2  Ehrlich,  Deut.  med.  Woch.,  1882;    Weigert,  Deut.  med.  Woch.,  1885. 

3  Ziehl,  Deut.  med.  Woch.,  1883;  Neelsen,  "  Lehrb.  d.  allg.  Path.,"  1894. 
»  GabbetL  Lancet,  1887. 


482  PATHOGENIC  MICROORGANISMS 

minutes,  at  the  end  of  which  time  all  elements  in  the  preparation  except 
the  acid-fast  bacilli  will  be  decolorized  and  counterstained. 

Tubercle  bacilli  in  very  young  culture  are  often  not  acid-fast  and  it 
is  not  always  possible  to  demonstrate  acid-fast  bacilli  in  pus  from  cold 
abscesses  in  sputum,  in  serous  exudates,  and  in  granulomatous  lesions 
of  the  lymph  nodes  which  can  be  shown  by  animal  inoculation  to  be 
tuberculous.  Much  ^  demonstrated  in  such  material  Gram-positive 
granules  which  lay  singly  in  short  chains  or  in  irregular  clumps,  and 
which  he  believed  to  be  non-acid-fast  tubercle  bacilli.  He  found  similar 
granules  in  cultures  of  tubercle  bacilli  which  showed  on  further  incuba- 
tion numerous  acid-fast  bacillary  forms.  His  work  has  been  repeatedly 
confirmed,  and  there  seems  little  doubt  but  that  these  granules  are  really 
tubercle  bacilli.  Their  demonstration  is  not,  however,  of  great  diag- 
nostic value,  as  other  bacilli  form  granules  of  the  same  appearance. 
Small  rods  and  splinters  are  also  found  which  stain  by  Gram's  method, 
but  not  by  carbol-fuchsin.^ 

To  find  "Much's  granules,"  smears  or  sections  are  steamed  in  a 
solution  of  methyl  violet  B.N.  (10  c.c.  of  saturated  alcohoHc  solution 
of  the  dye  in  100  c.c.  of  distilled  water  containing  2  per  cent  phenol). 
They  are  then  treated  with  Gram's  iodine  solution  1-5  minutes;  5  per 
cent  nitric  acid  1  minute;  3  per  cent  hydrochloric  acid  10  seconds;  ab- 
solute alcohol  and  acetone  equal  parts,  until  decolorized.  The  granules 
may  be  stained  by  other  modifications  of  Gram's  method.  Weiss  ^ 
has  devised  a  combination  stain.  One  part  of  Much's  methyl  violet 
is  mixed  with  three  parts  of  Ziehl's  carbol-fuchsin  and  filtered;  slides 
are  stained  for  24  to  48  hours  in  the  mixture.  They  are  then  decolorized 
as  in  Much's  method  and  counterstained  with  Bismarck  brown  or 
safranin  1  per  cent.  Both  acid-fast  and  Gram-positive  forms  are 
stained  by  this  method  and  in  the  red  may  be  seen  blue-black  granules. 

While  the  acid-fast  group  of  bacteria  is  composed  of  a  number  of 
organisms  to  be  mentioned  later,  a  few  only  of  these  offer  difficulties  of 
differentiation  from  the  tubercle  bacillus.  Those  to  be  considered 
practically  are  the  bacillus  of  leprosy  and  that  of  smegma.  The  latter 
bacillus,  because  of  its  distribution,  is  not  infrequently  found  to  ccgi- 
taminate  feces,  urine,  or  even  sputum,  and  it  is  important  to  apply  to 
suspected  specimens  one  or  the  other  of  the  stains  devised  for  the 
differentiation  of  the  smegma  bacillus  from  Bacillus  tuberculosis.     The 

1  Much,  Berl.  klin.  Woch.,  1908,  xlv,  700. 

2  Liebermeister,  Deutsche  med.  Woch.,  1909,  xxxv,  1324. 

3  Weiss,  Berl.  klin.  Woch.,  1909,  xlvi,  1797. 


THE  TUBERCLE  BACILLUS  483 

one  most  frequently  employed  is  that  of  Pappenheim.  ^  The  preparations 
are  stained  in  hot  carbol-fuchsin  as  before;  the  carbol-fuchsin  is  then 
poured  off  without  washing  and  the  preparation  immersed  in  a  solution 
made  by  saturating  a  1  per  cent  alcoholic  solution  of  rosolic  acid  with 
methylene-blue  and  adding  20  per  cent  of  glycerin.  In  such  prepara- 
tions tubercle  bacilli  remain  red,  smegma  bacilli  appear  blue. 

Stained  by  Gram,  tubercle  bacilli  retain  the  gentian-violet. 

When  tubercle  bacilli  are  very  sparsely  present  in  sputum  and 
other  material  it  may  be  impossible  to  find  them  by  direct  examination, 
and  often  the  only  method  of  finding  them  will  be  animal  inoculation. 
However,  a  number  of  methods  have  been  devised  by  which  the  bacilli 
may  be  concentrated  in  such  a  way  that  they  may  be  found  even  when 
a  few  only  are  present.  One  of  these  is  to  add  peroxide  of  hydrogen  to 
the  sputum.  By  this  the  mucus  is  dissolved  out  and  the  soUd  particles 
settle  or  may  be  centrifugalized.  A  method  very  commonly  employed 
to-day  is  that  which  depends  on  the  use  of  ''antiformin."  This  is  a 
preparation  used  extensively  for  the  cleansing  of  vats  in  breweries. 
It  is  described  by  Rosenau  ^  as  consisting  of  equal  parts  of  liquor  sodse 
chlorinatse  and  a  15  per  cent  solution  of  caustic  soda.  The  formula  for 
liquor  sodse  chlorinatee  he  gives  as: 

Sodium  carbonate 600 

Chlorinated  lime 400 

Distilled  water 4,000 

If  sputum  is  poured  into  a  10  to  15  per  cent  solution  of  antiformin 
and  allowed  to  stand  for  several  hours,  most  of  the  other  elements  of 
the  sputum,  cells,  and  bacteria,  will  dissolve  out,  and  acid-fast  baciUi 
be  left  in  the  residue.  Strangely  enough  they  are  not  killed  by  this- 
process  and  if  sufficiently  washed  may  be  cultivated  or  can  produce 
lesions  in  guinea-pigs. 

Isolation  and  Cultivation. — Tubercle  bacilli  are  not  easily  cultivated. 
Their  slowness  of  growth  precludes  isolation  by  plating.  The  first  isola- 
tions by  Koch  ^  were  made  upon  coagulated  blood  serum  from  tuber- 
culous tissue. 

Isolation  from  tuberculous  material  may  be  aided  by  inoculation 
into  guinea-pigs.     These  animals  will  withstand  the  acute  infection 

.-K^ 

i>h^  Pappenheim,  Berl.  klin.  Woch.,  1898. 

}•, -^  Rosenau,   "Preventive  Medicine  and  Hygiene,"   D.  Appleton,  N.  Y.,  1913; 
Uhlenhuth,  Berl.  klin.  Woch.,  No.  29,  1908. 
,  3  Kochf  loc.  cit. 


484  PATHOGENIC  MICROORGANISMS 

produced  by  the  contaminating  organisms  and  succumb  later  (four  to 
six  weeks)  to  tuberculosis.  The  bacilli  may  then  be  obtained  by  culti- 
vations from  lymph  nodes  or  other  foci  which  contain  only  tubercle 
bacilli.  When  isolation  from  sputum  is  attempted,  whether  directly 
or  by  means  of  animal  inoculation,  the  sputum  may  be  rendered  com- 
paratively free  from  contaminating  bacteria  by  washing.  The  sputum 
is  rinsed  in  running  water  to  free  it  from  pharyngeal  mucus.  It  is  then 
washed  in  eight  or  ten  changes  of  sterile  water.  The  material  selected 
is  taken  from  the  center  of  the  washed  mass,  if  possible  from  the  flakes 
of  caseous  material  visible  in  such  sputum. 

For  the  isolation  of  tubercle  bacilli  from  sputum  and  other  materials  in 
which  contaminating  bacteria  of  other  species  are  present,  Petroff  ^  has  devised 
an  excellent  method  which  has  been  tried,  out  and  used  with  success  in  our 
laboratory  by  Dr.  H.  R.  Miller.  The  principles  on  which  Petroff's  method 
rests  are,  first  of  all,  the  bactericidal  power  of  3%  sodium  hydroxid  on  non- 
acid-fast  bacteria,  and  the  selective  action  of  dyes  like  gentian  violet  on  bac- 
terial growth,  as  first  practically  utilized  by  Churchman  (See  page  140). 

The  medium  used  by  Petroff  is  made  as  follows: 

I.  Meat  Juice.  500  grams  of  beef  or  veal  are  infused  in  500  c.c.  of  a  15% 
solution  of  glycerin  in  water,  in  a  cool  place.  After  24  hours  the  meat  is  squeezed 
in  a  sterile  press  and  the  infusion  collected  in  a  sterile  beaker. 

II.  Eggs.  The  shells  of  the  eggs  are  sterilized  by  10  minute  immersion  in 
70%  alcohol.  They  are  broken  into  a  sterile  beaker,  well  mixed  and  filtered 
through  "sterile  gauze.  One  part  of  meat  juice  is  added  to  two  parts  of  egg  by 
volume. 

III.  Gentian  Violet.  1%  alcoholic  solution  of  gentian  violet  is  added  to 
make  a  final  proportion  of  1  :  10,000. 

The  three  ingredients  are  well  mixed.  The  medium  is  tubed  and  inspissated 
as  usual. 

Petroff  recommends  for  sputum  the  following  technique:  Equal  parts  of 
sputum  and  3%  sodium  hydroxid  are  shaken  and  incubated  at  38°  C.  for  15  to  30 
minutes,  the  time  depending  on  the  consistency  of  the  sputum.  The  mixture 
is  neutralized  to  litmus  with  hydrochloric  acid  and  centrifugalized.  The  sedi- 
ment is  inoculated  into  the  medium  described  above.  Pure  cultures  are  ob- 
tained in  a  large  proportion  of  cases. 

Petroff's  method  has  been  applied  by  him  to  feces,  in  which  the  problem 
is  made  more  difficult  by  the  presence  of  many  spore-formers  which  resist 
sodium  hydroxid.  Feces  is  collected  and  diluted  with  three  volumes  of  water, 
and  then  filtered  through  several  thicknesses  of  gauze.  The  filtrate  is  satu- 
rated with  sodium  chlorid  and  left  for  half  an  hour.    The  floating  film  of  bac- 

1  Petroff,  Johns  Hopkins  Hosp.  Bull.,  vol.  xxvi,  No.  294,  August,  1915,  p.  276. 


THE  TUBERCLE  BACILLUS  485 

teria  is  collected  in  a  wide-mouthed  bottle  and  an  equal  volume  of  normal 
sodium  hydroxid  is  added.  This  is  shaken  and  left  in  the  incubator  for  three 
hours,  shaking  every  half  hour.  It.  is  then  neutralized  to  litmus  with  normal 
hydrochloric,  centrifugalized,  and  the  sediment  planted. 

On  Mood  serum  at  37.5°  C,  colonies  become  visible  at  the  end  of 
eight  to  fourteen  days.  They  appear  as  small,  dry,  scaly  spots  with 
corrugated  surfaces.  After  three  or  four  weeks,  these  join,  covering 
the  surface  as  a  dry,  whitish,  wrinkled  membrane.  Coagulated  dog 
serum  is  regarded  by  Theobald  Smith  ^  as  a  favorable  media  for  the 
growth  of  tubercle  bacilli. 

Slants  of  agar,  to  which  whole  rahbWs  blood  has  been  added  in  quan- 
tities of  from  1  to  2  c.c.  to  each  tube,  make  an  excellent  medium. 

Cultivation  methods,  were  simplified  by  the  discovery  by  Roux  and 
Nocard  that  glycerin  facilitates  cultivation.  Upon  glycerin-agar  (gly- 
cerin 3  to  6  per  cent),  at  37.5°  C,  colonies  become  visible  at  the  end  of 
from  ten  days  to  two  weeks. 

Glycerin  houillon  (made  of  beef  or  veal  with  pepton  1%,  glycerin  six 
per  cent,  slightly  alkaline)  is  a  favorable  medium.  It  should  be  filled, 
in  shallow  layers,  into  wide-mouthed  flasks,  since  oxygen  is  essential. 
Transplants  to  this  medium  should  be  made  by  carefully  floating  flakes 
of  the  culture  upon  the  surface.  In  this  mediimi  the  bacilli  will  spread 
out  upon  the  surface,  at  first  as  a  thin,  opaque,  floating  membrane. 
This  rapidly  thickens  into  a  white,  wrinkled,  or  granular  layer,  spreading 
over  the  entire  surface  of  the  fluid  in  from  four  to  six  weeks.  Later, 
portions  of  the  membrane  sink.  In  old  cultures,  the  membrane  becomes 
yellowish.    These  cultures  emit  a  peculiar  aromatic  odor. 

Glycerin  potato  forms  a  favorable  culture  medium  for  the  bacillus. 

Hesse  ^  has  devised  a  medium  containing  a  proprietary  preparation 
known  as  ^'Nahrstoff  Heyden,"  upon  which  tubercle  bacilli  are  said  to 
proliferate  more  rapidly  than  other  bacteria.  His  method  has  yielded 
excellent  results.    It  is  prepared  as  follows: 

"Nahrstoflf  Heyden"' 10  grams 

Sodium  chlorid 5      " 

Glycerin. 30      " 

Agar 10      " 

Normal  sodium  solution 5  c.c. 

Aq.  dest 1,000    " 

1  Th.  Smith,  Jour.  Exp.  Med.,  iii,  1898. 

2  Hesse,  Zeit.  f.  Hyg.,  xxxi.  ^  "Nahrstoff  Heyden"  is  prepared  in  Germanyf 
It  is  a  white  powder  similar  to  nutrose. 


486 


PATHOGENIC  MICROORGANISMS 


Biological  Considerations. — The  tubercle  bacillus  is  dependent  upon 
the  access  of  oxygen.  Its  optimum  temperature  is  37.5°  C.  Tem- 
peratures below  30°  and  above  42°  C.  inhibit  its  growth.  In  fluid  media, 
the  bacilli  are  killed  by  60°  in  fifteen  to  twenty  minutes,  by  80°  in  five 

minutes,  by  90°  in  one  to  two  min- 
'  utes.  They  will  withstand  dry  heat 
at  100°  C.  for  one  hour.  They  are 
resistant  to  cold.  The  comparatively 
high  powers  of  resistance  of  the  bacil- 
lus are  attributed  to  the  protective 
qualities  of  the  waxy  cell  membrane.^ 
The  life  of  cultures,  kept  in  favor- 
able environment,  is  from  two  to  eight 
months,  varying  to  some  extent  with 
the  nature  of  the  culture  medium. 
The  viability  of  the  bacilli  in  sputum 
is  of  great  hygienic  importance.  In 
most  sputum  they  may  remain  alive 
and  virulent  for  as  long  as  six  weeks, 
in  dried  sputum  for  more  than  two 
months.^ 

Five  per  cent  carbolic  acid  kills 
the  bacilli  in  a  few  minutes.^  Used 
for  sputum  disinfection,  where  the 
bacilli  are  protected,  complete  disin- 
fection requires  five  to  six  hours.  Bi- 
chloride of  mercury  is  not  very  efficient 
for  sputum  because  of  the  formation 
of  albuminate  of  mercury.  For  room 
disinfection,  formaldehyde  gas  is  efficient.  Direct  sunlight  kills  in 
a  few  hours. 

Pathogenicity. — The  tubercle  bacillus  gives  rise  in  man  and  suscep- 
tible animals  to  specific  inflammation  which  is  so  characteristic  that  a 
diagnosis  of  tuberculosis  may  be  made  by  histological  examination, 
even  without  the  finding  of  tubercle  bacilli.  The  foci  known 
as  tubercles  have  been  studied  by  Baumgarten  ^  and  many  others 

^  Th.  Smith,  Jour.  Exper.  Med.,  1899;  Grancher  et  Ledoux-Lebard,  Arch,  de  med. 
exper;,  1892;  Galtier,  Compt.  rend,  de  I'acad.  des  sci.,  1887. 

2  Schell  und  Fischer,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 

3  De  Toma,  Ann.  di  med.,  1886.   .  *  Baumgarten,  Berl.  klin.  Woch.,  1901. 


Fig.  104. — Culture  of  Bacillus 
Tuberculosis  in  Flask  of  Glyc- 
erin Bouillon. 


THE  TUBERCLE  BACILLUS  48? 

and  descriptions  may  be  found  in  any  text-book  of  pathological 
anatomy. 

In  man,  tuberculosis  is  the  most  common  of  diseases.  Naegeli's  sta- 
tistics, based  on  a  large  series  of  autopsies,  show  not.  only  the  frequency 
of  the  disease,  but  its  relation  to  age.  Before  one  year  of  age,  he  finds  it 
very  rare.  From  the  first  to  the  fifth  year  it  is  rare,  but  usually  fatal. 
From  the  fifth  to  the  fourteenth  year,  one-third  of  his  cases  showed 
tuberculosis;  from  the  fourteenth  to  the  eighteenth  year,  one-half  of 
the  cases.  Between  eighteen  and  thirty,  almost  all  the  cases  examined 
showed  some  trace  of  tuberculous  infection.  Three-quarters  of  these 
were  active,  one-quarter  healed.  Two-fifths  of  all  deaths  occurring  at 
these  ages  were  due  to  tuberculosis.  After  the  age  of  thirty,  active 
lesions  gradually  diminished,  healed  lesions  increased. 

In  1900  Pry  or  ^  stated  that  the  average  yearly  mortality  from 
tuberculosis  in  New  York  amounted  to  6,000,  and  that  in  Manhattan 
alone  there  were  constantly  20,000  tuberculous  persons.  Cornet  ^  esti- 
mates that  in  1894  the  deaths  in  Germany  from  all  other  infectious 
diseases  amounted  to  116,705,  those  from  tuberculosis  alone  to  123,904. 
Similar  statistics  might  be  chosen  from  the  health  reports  of  any  large 
city.  While  the  disease  is  less  common  in  rural  districts  than  in  large 
towns,  the  difference  is  not  so  striking  as  is  generally  supposed. 

In  man,  pulmonary  infection  is  the  commonest  type.  Besides  this, 
tuberculous  processes  may  be  found  in  the  skin,  the  bones,  the  joints, 
the  organs  of  special  sense,  and  the  abdominal  viscera  and  peritoneum. 
No  part  of  the  human  body  is  exempt  from  the  danger  of  infection. 

Infection  in  man  may  take  place  by  inhalation,  through  the  skin 
or  the  digestive  apparatus.  V.  Behring  ^  has  expressed  the  belief  that 
a  large  percentage  of  all  cases  of  tuberculosis  originate  in  childhood 
from  infection  through  the  intestinal  tract.  He  determined  that 
tubercle  bacilli  may  penetrate  the  intestinal  mucosa  without  causing 
lesions.  Behring' s  contention  has  caused  a  great  deal  of  discussion, 
and  the  question  he  has  raised  is  intimately  bound  up  with  the  problem 
of  the  virulence  of  bovine  tubercle  bacilli  for  human  beings,  as  he  as- 
sumes that  the  infection  is  due  to  the  use  of  infected  milk. 

The  problem  is  plainly  of  the  greatest  importance,  and  for  this 
reason  has  been  diligently  investigated  during  the  last  few  years.  The 
only  reliable  method  of  approaching  it  has  been  to  isolate  the  tubercle 

1  Pry(yr,  Med.  News,  Ixxvii,  1900. 

«  C(ymet,  "Die  Tuberculose,"  Wien,  1899,  p.  1. 

»  V,  Behring,  Deut.  med.  Woch.,  39,  1903. 


488 


PATHOGENIC  MICROORGANISMS 


bacilli  from  diseased  human  beings  and  determine  for  each  case  whether 
the  guilty  organism  belonged  to  the  human  or  bovine  type.  These 
types  can  be  differentiated  definitely  by  cultural  characteristics  and 
pathogenicity,  and  it  is  not  likely  that  the  type  changes  during  the 
sojourn  in  the  human  body.  Granted  this  permanence  of  type,  it  is 
naturally  of  much  value  in  revealing  the  source  of  an  infection,  to  de" 

Combined  Tabulation,  Cases  Reported  and  Own  Series  of  Cases 
(From  Park  and  Krumwiede,  loc.  cit.) 


Diagnosis. 


Adults 
16  Years 
and  Over, 


Human   Bovine 


Children 
5  to  16  Years. 


Human    Bovine 


Children 
Under  5 
Years. 


Human    Bovine 


Pulmonary  tuberculosis 

Tuberculous  adenitis,  axillary  or  inguinal 

Tuberculous  adenitis,  cervical 

Abdominal  tuberculosis 

Generalized  tuberculosis  alimentary  origin . . . 

Generalized  tuberculosis 

Generalized  tuberculosis,  including  meninges, 

alimentary  origin 

Generalized  tuberculosis,  including  meninges . 

Tuberculous  meningitis. 

Tuberculosis  of  bones  and  joints 

Genito-urinary  tuberculosis 

Tuberculosis  of  skin 

Miscellaneous  Cases: 

Tuberculosis  of  tonsils 

Tuberculosis  of  mouth  and  cervical  nodes 

Tuberculous  sinus  or  abscesses 

Sepsis,  latent  bacilli 

Totals 


568 

2 

22 

15 

6 

28 


18 
11 

1 


2 
677 


20 

7 
3 

1 


11 

4 
33 

7 
2 
4 

1 

7 
2 
26 
1 
1 


99        33 


12 
2 

15 
6 

13 

28 

3 
45 
14 
21 


1 
161 


20 

13 

10 

5 

8 

1 
2 


59 


Mixed  or  double  infections,  4 


termine  whether  or  not  a  human  being  is  harboring  a  bacillus  of  the 
human  type  or  one  of  the  bovine  type. 

One  of  the  most  valuable  contributions  made  to  this  problem  during 
the  last  three  years  is  that  of  Park  and  Krumwiede.^  The  above 
tabulation  is  taken  from  their  paper  and  represents  a  summary  of  their 
own  cases  and  those  reported  by  others. 


*  Park  and  Krumwiede,  Jour,  of  Med.  Res.,  Oct.,  1910. 


THE  TUBERCLE  BACILLUS  489 

From  this  table  it  is  evident  that  out  of  a  total  of  1,042  cases,  101 
only  were  bovine  in  origin  and  over  50  per  cent  of  these  occurred  in 
children  under  five  years  of  age.  Fifty-one  out  of  the  59  cases  occurring 
in  the  161  infants  were  directly  or  indirectly  traced  to  alimentary  in- 
fection. 

It  seems  reasonably  accurate,  therefore,  to  state  the  case  as  follows: 
Human  adults  are  relatively  insusceptible  to  bovine  infection.  Such 
infection  can  take  place,  but  is  unusual.  Below  16  years  of  age  the 
human  race  is  relatively  more  susceptible  and  up  to  this  age  the  danger 
of  milk  infection  is  unquestionably  great,  this  source  accounting  for 
about  one-third  of  the  cases.  Below  5  years  the  danger  is  greatest. 
This  table  alone  should  form  sufficient  evidence  to  silence  absolutely 
any  doubts  as  to  the  dangers  of  milk  infection  and  remove  any  objections 
to  the  most  rigid  sanitary  control  of  milk  supplies. 

On  the  other  hand,  it  also  shows  that  Behring's  original  claims  were 
far  too  sweeping  and  can  not  be  upheld. 

Rosenberger  ^  has  recently  reported  finding  tubercle  bacilli  in  the 
circulating  blood  of  all  cases  of  human  tuberculosis  which  he  examined. 
This  announcement  aroused  much  interest  and  has  led  to  many  investi- 
gations by  other  workers.  Rosenberger's  results  were  obtained  by  mor- 
phological examination  of  smears  of  citrated  blood  taken  from  the 
patients,  dried  upon  slides  and  laked  with  distilled  water.  Many  other 
observers  have  failed  to  confirm  Rosenberger's  results.  Anderson  ^  ex- 
amined 47  cases  in  which  tubercle  bacilli  were  found  in  the  sputum  and  one 
case  of  joint  tuberculosis.  In  none  of  these  48  cases  was  he  able  to  obtain 
tubercle  bacilli,  either  by  morphological  examination  nor  by  guinea-pig 
inoculation.  Brem  ^  subsequently  found  that  laboratory  distilled  water 
may  frequently  contain  acid-fast  saprophytes — a  fact  which  may 
account  in  many  cases  for  errors  when  morphological  examination  alone 
is  relied  upon  and  blood  examined  by  the  technique  of  Rosenberger. 
This,  too,  is  suggested  by  the  finding  of  acid-fast  bacilli  in  the  blood  of 
perfectly  healthy  individuals.  Therefore,  although  the  bacilli  may  be 
present  in  the  blood  in  a  certain  number  of  cases  it  does  not  seem  likely 
that  they  are  so  distributed  in  anything  like  the  high  percentages  found 
by  Rosenberger.'* 

Bacillus  tuberculosis   (typus  humanus)   is  pathogenic  for  guinea- 

1  Rosenberger,  Am.  Jour,  of  Med.  Sc,  cxxxvii,  1909. 

2  Anderson,  U.  S.  P.  H.  Service,  Hygienic  Lab.,  Bull.  57,  1909. 

3  Brem,  Jour.  A.  M.  A.,  liii,  1909. 

^Suzuki  and  Takaki,  CentralbL  f.  Bakt.^  Ixij  1911. 


490  PATHOGENIC  MICROORGANISMS 

pigs,  less  markedly  for  rabbits,  and  still  less  so  for  dogs.  It  is 
slightly  pathogenic  for  cattle,  a  question  spoken  of  more  extensively 
below. 

Chemical  Analysis  of  Tubercle  Bacilli.^ — Diligent  efforts  by  many 
investigators  to  isolate  the  specific  toxins  which  lend  tubercle  bacilli 
their  pathogenic  properties  have  led  to  careful  chemical  analysis  of  the 
organisms.  About  85.9  per  cent  of  the  bacillus  consists  of  water;  20 
to  26  per  cent  of  the  residue  can  be  extracted  with  ether  and  alcohol. 
This  material  consists  of  fatty  acids  and  waxy  substances  (fatty  acids 
in  combination  with  the  higher  alcohols).  The  residue  after  alcohol- 
ether  extraction  is  composed  chiefly  of  proteids.  These  can  be  extracted 
with  dilute  alkaline  solutions,  and  consist  chiefly  of  nucleo-albumins. 
A  nuclein  present  in  this  fraction  shows  extremely  high  toxicity  and 
has,2  therefore,  been  suspected  of  being  the  pathogenic  principle  of  the 
bacillus.  After  these  extractions  the  remainder  contains  ''cellulose," 
supposed  to  represent  the  framework  of  the  cell  membrane,  and  an  ash 
rich  in  calcium  and  magnesium. 

Toxins  of  the  Tubercle  Bacillus. — The  Tuberculins. — Filtrates  of 
bouillon  cultures  of  Bacillus  tuberculosis  ^  will  occasionally  produce 
slight  emaciation  when  injected  into  guinea-pigs,  and  when  administered 
to  tuberculous  subjects  in  sufficient  quantity  will  give  rise  to  marked 
increase  of  temperature.  It  is  likely,  therefore,  that  the  tubercle 
bacillus  actually  secretes  a  soluble  toxin.^ 

The  chief  toxic  principles,  however,  of  Bacillus  tuberculosis  are 
probably  endotoxins  or  bacterial  proteins,  bound  during  cell  life  to  the 
body  of  the  bacillus.  Dead  bacilli  will  produce  sterile  abscesses  when 
injected  into  animals.  Prudden  and  Hodenpyl,^  Straus  and  Gamaleia,® 
and  others,^  moreover,  have  shown  that  the  injection  of  dead  and  care- 
fully washed  cultures  of  this  bacillus  will  produce  lesions  histologi- 
cally similar  to  those  occurring  after  infection  with  the  living  germs, 
and  will  often  lead  to  marasmus  and  other  systemic  symptoms  of 
poisoning. 

The  hope  of  actively  immunizing  with  substances  obtained  from 

^  Hammer schlag,  Cent.  f.  kKn.  Med.,  1891;  Weyl,  Deut.  med.  Woch.,  1891;  De 
Schweinitz  and  Dorset,  Jour.  Amer.  Chem.  Soc,  1895;  Hammerschlag,  loc.  cit. 
2  Behnng,  Berl.  klin.  Woch.,  1899. 
^Straus  and  Gamdleia,  Arch.  med.  exp.,  1891. 
*  Denys,  "Le  Bouillon  Filtre,"  Louvain,  1905. 

^  Prudden  and  Hodenpyl,  N.  Y.  Med.  Jour.,  June,  1S91;  Prudden,  ibid.,  Deo.  5.. 
^  Straus  and  Gamaleia,  loc.  cit. 
7  Majucd,  Cent,  f .  allg.  Path.,  1890. 


THE  TUBERCLE  BACILLUS  491 

dead  bacilli  led  Koch  to  employ  various  methods  of  extraction  of  cultures 
for  the  manufacture  of  tuberculin. 

''Old  Tuberculin*''^  (Koch)  ("T.A.K.")-— The  first  tuberculin  made  by 
Koch  is  produced  in  the  following  manner:  Tubercle  baciUi  are  grown 
in  slightly  alkaline  5  per  cent  glycerin-pepton  bouillon  for  six  to  eight 
weeks.  At  the  end  of  this  time,  growth  ceases  and  the  corrugated 
peUicle  of  tubercle  bacilli,  which  during  growth  has  floated  on  the 
surface,  begins,  here  and  there,  to  sink  to  the  bottom.  The  entire 
culture  is  then  heated  on  a  water-bath  at  about  80°  C,  until  reduced  to 
one-tenth  of  its  original  volume.  It  is  then  filtered  either  through 
sterile  filter  paper  or  through  porcelain  filters.  The  resulting  filtrate  is 
a  rich  brown,  syrupy  fluid,  containing  the  elements  of  the  original  cul- 
ture medium  and  a  50  per  cent  glycerin  extract  of  the  tubercle  bacilli. 
While  the  glycerin  is  of  sufficient  concentration  to  preserve  it  indef- 
initely, 0.5  per  cent  phenol  may  be  added  as  an  additional  precaution. 
Dilutions  of  this  fluid  are  used  for  diagnostic. and  therapeutic  purposes. 

"New  Tuberculin''  ^  (Koch)  (TA,  TO,  TR).— Koch  believed  that 
the  immunity  resulting  from  treatment  with  the  old  tuberculin  was 
purely  an  antitoxic  immunity,  devoid  of  all  antibacterial  action.  The 
use  of  whole  dead  tubercle  bacilli  for  immunization  purposes,  however, 
was  impracticable;  because,  injected  subcutaneously,  they  were  not 
absorbed,  and  introduced  intravenously  they  were  deposited  in  the  lungs 
and  gave  rise  to  lesions.  Koch  was  led,  therefore,  to  resort  to  more 
energetic  extraction  of  the  bacilli  in  the  hope  of  procuring  a  substance 
which  could  be  easily  absorbed  and  would  at  the  same  time  give  rise, 
when  injected,  to  antibodies  more  definitely  bactericidal.  By  extract- 
ing tubercle  bacilli  with  decinormal  NaOH,  for  three  days,  filtering 
through  paper  and  neutralizing,  he  obtained  his  TA  (alkahne  tubercu-. 
lin).  This  preparation  seemed  to  fulfil  some  of  the  hopes  of  its  dis- 
coverer, but  had  the  disadvantage  of  often  producing  abscesses  at  the 
points  of  injection.  Koch  then  resorted  to  mechanical  trituration  of 
the  bacilli.  The  method  he  subsequently  followed  for  tuberculin  pro- 
duction is  now  extensively  used,  and  is  carried  out  as  follows:  ^ 

Virulent  cultures  of  tubercle  bacilli  are  dried  in  vacuo  and  thoroughly 
ground  in  a  mortar.  Grinding  is  continued  until  stained  preparations 
reveal  no  intact  bacilli.  (This  is  done  by  machinery  in  all  large  manu- 
factories.) One  gram  of  the  dry  mass  is  shaken  up  in  100  c.c.  of  sterile 
distilled  water.     This  mixture  is  then  centrifugalized  at  high  speed. 

1  Koch,  Cent.  f.  Bakt.,  1890;  Deut.  med.  Woch.,  1891. 

2  Koch,  Deut.  med.  Woch.,  14,  1897.  ^  Ruppel,  Lancet,  March  28,  1908. 

32 


492  PATHOGENIC  MICROORGANISMS  i 

The  supernatant  fluid,  known  as  TO  (Tuberculin-Oberschicht),  contains 
the  water-soluble  constituents  of  the  bacillus,  gives  no  precipitate  on 
the  addition  of  50  per  cent  glycerin,  and  has  the  'same  physiological 
action  as  the  old  tuberculin.  The  residue  TR  (TubercuUn-Ruckstand), 
after  pouring  off  TO,  is  again  dried  and  ground  up,  and  again  shaken  in 
water  and  centrifugalized.  This  process  is  repeated  several  times, 
and  eventually,  after  three  or  four  repetitions,  all  the  TR  goes  into 
emulsion.  The  total  volume  of  water  used  for  these  TR  extractions 
should  not  exceed  100  c.c.  All  of  the  TR  emulsions  are  then  mixed  to- 
gether. This  gives  TR  a  precipitate  with  50  per  cent  of  glycerin,  and  is 
supposed  by  Koch  to  contain  substances  important  in  producing  an 
antibacterial  immunity.  For  purposes  of  standardization,  the  amount 
of  solid  substance  in  5  c.c.  of  the  TR  is  determined  by  evaporation  in 
vacuo  and  drying.  To  the  rest  are  added  a  little  glycerin  and  formalde- 
hyde and  enough  water  to  allow  each  cubic  centimeter  of  the  solution 
to  contain  0.002  grams  of  solid  material.  Thus  the  culture  and  the 
medium  remaining  the  same,  fairly  accurate  standardization  is  possible. 

''New  TubercuUn-Bacillary  Emulsion.''  ^ — In  1901,  Koch  combined 
*'T0"  and  "TR"  by  putting  forth  a  preparation  referred  to  as 
"Bazillenemulsion."  This  consists  of  an  emulsion  of  pulverized  bacilli 
1  :  100  in  distilled  water.  After  several  days  of  sedimentation  to  re- 
move the  coarser  particles,  the  supernatant  fluid  is  poured  off  and  fifty 
per  cent  volume  of  glycerin  is  added  to  it  for  purposes  of  pi'eservation. 
This  preparation  contains  5  milligrams  of  solid  substance  in  each  cubic 
centimeter. 

Bouillon  Filtre  (Denys).^ — This  preparation  consists  of  the  filtrate 
(through  Chamberland  filters)  of  5  per  cent  glycerin-pepton-bouillon 
cultures  of  Bacillus  tuberculosis.  Phenol  0.25  per  cent  is  added  to 
insure  sterility.  The  filtered  bouillon  corresponds  to  the  unconcentrated 
old  tuberculin  of  Koch,  but,  not  having  been  heated,  is  supposed  by 
Denys  to  contain  important  soluble  and  possibly  thermolabile  secretory 
products  of  the  bacillus. 

Tuberculoplasmin  (Buchner  and  Hahn).^ — Buchner  and  Hahn,  by 
crushing  tubercle  bacilli  by  subjecting  them  to  a  pressure  of  400 
atmospheres,  obtained  a  cell-juice  in  the  form  of  an  amber  fluid,  to 
which  they  attributed  qualities  closely  analogous  to  those  of  TR. 


1  Koch,  Deut.  med.  Woch,,  1901. 

2  Denys,  "Le  Bouillon  Filtre,"  Louvain,  1905. 

'  Buchner  und  Hahn,  Munch,  med.  Woch.,  1897;  Hahn,  ibid 


THE  TUBERCLE  BACILLUS  493 

Other  tuberculins  are  those  of  Beraneck/  highly  recommended 
clinically  by  Sahli,^  that  of  Klebs,^  and  the  tuberculin  produced  from 
bovine  tubercle  bacilli  by  Spengler.'^ 

Diagnostic  Use  of  Tuberculin. — Subcutaneous  Use. — The  preparation 
usually  employed  for  diagnostic  purposes  is  Koch's  "Old  Tuberculin'* 
(Alttuberculin).  This  preparation  is  administered  by  hypodermic  injec- 
tion of  small  quantities  obtained  by  means  of  dilutions.  The  dilutions 
are  best  made  with  a  0.5  per  cent  aqueous  carboUc  acid  solution.  In 
practice  a  1  per  cent  solution  is  made  by  pipetting  0.1  c.c.  of  tuberculin 
into  9.9  c.c.  of  the  0.5  per  cent  carbolic  solution.  A  cubic  centimeter 
of  this  then  contains  0.01  c.c.  of  tuberculin.  One  c.c.  of  this  solution 
added  to  9  c.c.  of  0.5  per  cent  carboUc  acid  gives  a  solution  in  whieh 
each  cubic  centimeter  contains  0.001  c.c,  or  1  milligram  of  tuberculin. 

The  initial  dosage  in  adults  in  Koch's  ^  early  work,  and  as  used  by 
Beck  ^  on  a  large  number  of  patients,  was  1  milligram.  This,  according 
to  present  opinions,  is  too  high,  and  most  clinicians  to-day  prefer  0.1 
to  0.2  of  a  milligram.  If  after  three  or  four  days  no  reaction  has  occurred, 
a  second  dose  of  1  milligram  is  given. 

The  reaction  itself  is  recognized  chiefly  by  the  changes  in  tem- 
perature. In  a  positive  reaction  the  patient's  temperature  will  begin 
to  increase  within  six  to  eight  hours  after  injection,  rising  sharply 
within  a  few  hours  to  0.5  or  1.5°  higher  than  the  temperature  before  in- 
jection. It  then  sinks  more  gradually  than  it  rose,  the  reaction  usually 
being  complete  within  thirty  to  thirty-six  hours.  "With  the  temperature 
there  may  be  nausea,  a  chill,  rapid  pulse;  and  general  malaise.  Locally 
visible  tuberculous  processes,  such  as  lupus,  lymph  nodes,  etc.,  may 
become  more  tender  or  swollen,  and  if  the  tuberculosis  is  pulmonarjr, 
there  may  be  coughing  and  increased  expectoration.  The  temperatures 
of  persons  subjected  to  the  test  should  be  taken  regularly  for  three  or 
four  days  before  tuberculin  is  used. 

Ophthalmo-TuhercuUn  Reaction. — "Wolff-Eisner^  and,  soon  after 
him,  Calmette,^  proposed  a  method  of  using  tuberculin  for  diagnostic 

*  Beraneck,  Compt.  rend,  de  I'acad.  des  scL,  1903. 

2  Sahli,  Corrbl.  d.  Schw.  Aerzte,  1906. 

3  Kiehs,  Cent.  f.  Bakt.,  1896;  Deut.  med.  Woch.,  1907. 

*  Spengler,  Deut.  med.  Woch.,  xxxi,  1904;  xxxi  and  xxxiv,  1905. 

6  Koch,  Deut.  med.  Woch.,  1890. 

«  Beck,  Deut.  med.  Woch.,  1899.  • 

7  Wolff-Eisner,  Berl.  med.  Gesell.,  May  15,  1907. 

*  Calmette,  Acad,  des  sci.,  June  17,  1907.  t 


494  PATHOGENIC  MICROORGANISMS 

purposes  by  instillation  into  the  conjunctival  sac.  In  tuberculous 
patients  this  process  is  followed  by  a  sharp  conjunctival  congestion 
lasting  from  one  to  several  days. 

The  preparation  used  for  this  purpose  is  produced  in  the  following 
way: 

**01d  Tuberculin"  is  treated  with  double  the  quantity  of  95  per 
cent  alcohol,  and  the  precipitate  allowed  to  settle  and  the  alcohol  then 
filtered  off  through  paper.  The  sediment  is  washed  with  70  per  cent 
alcohol  until  the  filtrate  runs  clear,  then  pressed  between  layers  of 
filter  paper  to  remove  excess  of  moisture,  scraped  into  a  dish,  dried 
in  vacuo  over  H2SO4,  and  broken  up  in  a  mortar  under  a  hood. 

Solutions  of  the  powder  are  made  in  sterile  normal  salt  solution,  1 
per  cent  by  weight,  boiled  and  filtered.  The  solutions  are  used  in 
strengths  of  0.5  to  1  per  cent,  a  drop  of  which  is  instilled  into  the  con- 
junctival sac.^ 

Cutaneous  Tuberculin  Reaction. — ^Von  Pirquet  ^  has  suggested  the 
cutaneous  use  of  tuberculin  for  diagnostic  purposes.  A  25  per  cent 
solution  of  *'01d  Tuberculin"  was  first  used.  At  present  the  undi- 
luted substance  is  employed. 

After  sterilization  of  the  patient's  forearm,  two  drops  of  this  solu- 
tion are  placed  upon  the  skin  about  6  cm.  apart.  Within  each  of  these 
drops  scarification  is  done,  and  the  skin  between  them  sacrificed  as  a 
control.  Within  twenty-four  to  forty-eight  hours,  in  tuberculous 
patients,  erythema,  small  papules,  and  herpetiform  vesicles  will  ap- 
pear. According  to  recent  investigations,  about  70  per  cent  of  adults 
show  a  positive  reaction.    This  reverses  its  diagnostic  value  for  adults. 

Moro  ^  has  modified  this  by  simply  making  a  50  per  cent  ointment 
of  tuberculin  in  lanolin  and  rubbing  it  into  the  skin  without  scari- 
fication. 

Complement  Fixation  in  Tuberculosis.^ — The  problem  of  comple- 
ment fixation  in  tuberculosis  for  diagnostic  purposes  has  been  very 
actively  investigated  of  recent  years.  The  most  promising  results  have 
been  reported  with  an  antigen  made  by  Besredka  of  a  filtrate  of  an 
egg-meat-broth,  upon  which  culture  the  tubercle  bacilli  had  been  grown 
for  several  weeks ;  a  similar  filtrate  of  cultures  on  a  watery  extract  of 

1  Method  in  use  at  Saranac  and  kindly  communicated  by  Dr.  Baldwin. 

2  V.  Pirquet,  Berl.  klin.  Woch.,  xx,  1907;  Med.  Klinik,  xl,  1907. 
»  Moro,  Munch,  med.  Woch.,  1906,  p.  216. 

*  A  review  of  complement  fixation  tests  in  tuberculosis  will  be  found  in  an  article 
by  H.  R.  Miller,  Jour.  I  ab.  &  Clin.  Med.,  1916,  i,  816. 


THE  TUBERCLE  BACILLUS  495 

potato  with  glycerin,  used  by  Petroff ;  and  an  antigen  made  by  Miller 
and  Zinsser  by  triturating  dead  tubercle  bacilli  with,  dry  crystals  of 
NaCl  and  adding  distilled  water  to  isotonieity.  Craig,  Bronfenbren- 
ner  and  the  above-named  writers  have  reported  good  results  with 
these  various  antigens,  and,  although  too  early  to  say  which  will  prove 
most  useful,  it  is  clear  that  complement  fixation  methods  can  aid  in 
the  diagnosis  of  active  tuberculosis.  We  can,  of  course,  judge  con- 
cisely only  of  the  metliod  used  in  our  laboratory,  where  Miller  has 
followed  carefully  a  considerable  number  of  cases  on  which  the 
method  has  been  used.  It  would  appear  at  present  that  between  80 
and  90  per  cent  of  the  fixation  results  correspond  accurately  with 
clinical  findings. 

The  Tuberculin  Test  as  Applied  to  Cattle. — In  cattle,  the  symptoms 
of  tuberculosis  are  not  easily  detected  by  methods  of  physical  diag- 
nosis until  the  disease  has  reached  an  advanced  stage.  In  conse- 
quence, cows  may  be  elements  of  danger  without  appearing  in  any 
way  diseased  to  those  who  handle  them.  In  consequence,  routine 
examination  of  herds  by  the  tuberculin  test  has  become  one  of 
the  necessary  measures  in  'public  sanitation.  According  to  Mohler,^ 
an  accurate  diagnosis  may  be  established  in  at  least  97  per  cent  of  the 
cases.  It  is  natural  that  a  good  deal  of  objection  to  the  test  is  encoun- 
tered on  the  part  of  dairy  farmers  and  cattle  raisers,  and  recently  it  has 
been  publicly  claimed  that  the  cattle  are  injured  by  the  test.  There  is, 
however,  no  scientific  basis  for  this  belief,  if  the  test  is  carried  out  care- 
fully and  intelligently.  As  a  matter  of  fact,  the  systematic  use  of  the 
test  would  eventually  be  distinctly  advantageous  to  the  owners  of  the 
cattle  themselves,  since  it  has  been  shown  that  cows,  even  in  the  early 
stages  of  the  disease,  may  expel  tubercle  bacilli,  either  during  respira-' 
tion  or  in  the  feces,  and  thus  become  a  menace  to  healthy  members  of 
the  herd. 

The  tuberculin  test  on  cattle  should  be  made  as  follows:  (The 
directions  given  below  are  taken  directly  from  the  circular  sent  out  from 
the  Bureau  of  Animal  Industr>^  at  Washington.) 

1.  Begin  to  take  the  rectal  temperature  at  6  a.m.,  and  take  it  every 
two  hours  thereafter  until  midnight. 

2.  Make  the  injection  at  midnight. 

3.  Begin  to  take  the  temperature  next  morning  at  6  a.m.,  and  con- 
tinue as  on  preceding  day. 

To  those  who  have  large  herds  to  examine,  or  are  unable  to  give  the 

1  MoAZer,.  Pub.  H.  and.Mar.  Hosp.  Serv.  Bull.  41,.  1908.. 


496  PATHOGENIC  MICROORGANISMS 

time  required  by  the  above  directions,  the  following  shortened  course  is 
recommended : 

1.  Begin  to  take  the  temperature  at  8  a.m.,  and  continue  every  2 
hours  until  10  p.m.  (omitting  at  8  p.m.,  if  more  convenient);  or  take  the 
temperature  at  8  a.m.,  12  m.,  and  10  P.M. 

2.  Make  the  injection  at  10  p.m. 

3.  Take  the  temperature  next  morning  at  6  or  8  a.m.,  and  every  2 
hours  thereafter  until  6  or  8  p.m. 

Each  adult  animal  should  receive  2  c.c.  of  the  tuberculin  as  it  is  sent 
from  the  laboratory.  (The  tuberculin  sent  out  from  the  central  labora- 
tory at  Washington  is  already  diluted;  2  c.c.  represents  0.25  c.c.  of  the 
concentrated  ''Old  Tuberculin''  of  Koch.)  Yearlings  and  two-year-olds, 
according  to  size,  should  receive  from  1  to  1.5  cubic  centimeters.  Bulls 
and  very  large  animals  may  receive  three  cubic  centimeters.  The  injec- 
tion should  be  made  beneath  the  skin  of  the  neck  or  shoulders  behind 
the  scapula,  after  washing  the  area  with  a  weak  carbolic  acid  solution. 

There  is  usually  no  marked  local  swelling  at  the  seat  of  the  injection. 

There  are  now  and  then  uneasiness,  trembling,  and  the  more  fre- 
quent passage  of  softened  dung.  There  may  also  be  sHght  acceleration 
of  the  pulse  and  of  the  breathing. 

The  febrile  reaction  in  tuberculous  cattle  following  the  subcutaneous 
injection  of  tuberculin  begins  from  six  to  ten  hours  after  the  injection, 
reaches  the  maximum  nine  to  fifteen  hours  after  the  injection,  and 
returns  to  normal  eighteen  to  twenty-six  hours  after  the  injection. 

A  rise  of  two  or  more  degrees  Fahrenheit  above  the  maximum  tem- 
perature observed  on  the  previous  day  should  be  regarded  as  an  indica- 
tion of  tuberculosis.  For  any  rise  less  than  this  a  repetition  of  the 
injection  after  four  or  six  weeks  is  highly  desirable. 

It  is  hardly  necessary  to  suggest  that  for  the  convenience  of  the  one 
making  the  test  the  animals  should  not  be  turned  out,  but  fed  and 
watered  in  the  stable.  It  is  desirable  to  make  note  of  the  time  of  feed- 
ing and  watering  and  of  any  temperature  fall  after  watering. 

The  tuberculin  should  not  be  used  later  than  six  weeks  after  the 
date  on  the  bottle,  nor  if  there  is  a  decided  clouding  of  the  solution. 

Therapeutic  Uses  of  Tuberculin. — Tuberculin  was  first  used  therapeu- 
tically, shortly  after  its  discovery,  by  Koch.^  Hailed  with  the  most 
optimistic  enthusiasm,  its  possibilities  were  overestimated  and  hope- 
less cases  were  treated  unskilfully,  with  unsuitable  dosage.  The  conse- 
quence was  that  harm  was  done,  the  method  was  attacked  by  Virchow 

1  Koch,  Deut.  med.  Woch.,  iii,  imh 


'tttti  ITTBERCLE  BACILLUS  49^ 

and  others  and  the  new  therapy  fell  into  almost  complete  neglect.  At 
present,  the  use  of  tuberculin  has  again  been  revived,  but  with  greater 
caution  and  with  a  thorough  understanding  of  its  limitations.  The 
tendency  has  been  toward  smaller  dosage  and  the  limitation  of  the  agent 
to  early  cases.  No  two  institutions  use  tuberculin  in  exactly  the  same 
manner,  and  it  is,  therefore,  impossible  to  do  more  than  outline  the 
general  scheme  of  treatment.  It  must  never  be  forgotten,  however, 
that  all  forms  of  tuberculin  treatment  consist  in  an  "active  immuniza- 
tion" in  which,  for  the  time  being,  the  toxemia  of  the  patient  is  increased 
rather  than  neutralized.  It  is  obvious,  therefore,  that  only  such  cases 
are  at  all  suitable  for  treatment  in  which  the  process  is  not  a  very  acute 
one.  The  general  principle  of  modern  tuberculin  therapy  seems  to  lie 
in  choosing  doses  so  small  that  no  marked  general  reaction  shall  follow. 
The  preparations  most  frequently  employed  are  Koch's  "Alttuber- 
cuHn,"  his  "TR,"  his  "Neu  Tuberkulin-Bazillen  Emulsion,"  and  the 
Bouillon  filtre  of  Denys.  Initial  doses  of  Alttuberculin  range  from  0.1 
to  0.01  of  a  milligram.  In  case  of  successful  avoidance  of  a  reaction, 
the  injection  may  be  repeated,  gradually  increasing,  about  twice  a  week. 
The  occurrence  of  a  reaction  should  be  the  signal  for  a  longer  interval 
and  a  slower  advance  in  the  size  of  the  dose. 

The  initial  dose  of  "TR"  is,  as  advised  by  Koch,i  about  0.002 
mgm.  This  usually  causes  no  reaction.  The  dose  is  doubled,  at  reason- 
able intervals,  up  to  1  mgm.  After  this,  further  increase  is  care- 
fully gauged  by  the  clinical  indications.  The  maximum  dose  is  about 
20  mgm. 

"Neu  Tuberkulin-Bazillen  Emulsion,"  ^  is  begun  with  a  dose  of  0.001 
mgm.  Gradual  increase  as  with  the  other  preparations  is  then  prac- 
ticed.   The  maximum  dose  is  about  10  mgm. 

Bouillon  filtre  has  been  used  chiefly  by  Denys  ^  and  with  apparently 
excellent  results.  Denys  is  very  Emphatic  in  advising  the  absolute 
avoidance  of  any  reaction.  He  begins  with  a  millionth  or  even  the 
tenth  of  a  millionth  of  a  cubic  centimeter  of  the  bouillon  and  in- 
creases with  extreme  caution.  His  dilutions  are  made  with  glycerin 
broth. 

Passive  Immunization  in  Tuberculosis. — ^Numerous  attempts  have 
been  made  to  immunize  tuberculous  subjects  with  the  sera  of  actively 

1  Koch,  Deut.  med.  Woch.,  xiv,  1897. 

^Bandelier  und  Roepke,  ''Lehrb.  d.  spezifisch.  Tub.  Ther.,"  Wurzburg,  1908; 
Koch,  Deut.  med.  Woch.,  1901. 

^  Denys,  "Le  Bouillon  filtre,"  Louvain,  190.5. 


498  PATHOGENIC   MICROORGANISMS 

immune  animals.  The  most  widely  used  method  of  producing  such 
serum  is  that  of  Maragliano. 

Maragliano^s  Serum} — Maragliano  believes  that  a  toxalbumin  is 
present  in  tubercle-bacillus  cultures  which  is  destroyed  by  the  heating 
employed  in  the  usual  tuberculin  production.  He  procures  this  sub- 
stance by  filtration  of  unheated  cultures  and  precipitation  with  alcohol 
(tossina  praecipitata) .  He  furthermore  makes  an  aqueous  extract  of 
the  bacillary  bodies.  With  these  two  substances  he  immunizes  horses. 
He  draws  blood  from  these  after  four  to  six  months  of  treatment.  The 
serum  is  extensively  used  in  Italy.  Its  value  is,  at  present,  very 
doubtful. 

Marmorek^s  Serum.^ — Marmorek  claims  that  the  poisons  produced  by 
Bacillus  tuberculosis  depend  largely  upon  the  medium  on  which  it  is 
grown.  He  advanced  the  view  in  1903  that  the  substances  obtained 
in  tuberculin  were  not  the  true  toxins  of  the  tubercle  bacillus,  that  there 
was  a  marked  difference  between  these  and  the  poisons  elaborated  by  a 
younger  (primitive)  phase  of  the  bacillus  as  it  occurs  only  within  the 
animal  body  or  on  media  composed  of  animal  tissue.  He  consequently 
grows  his  cultures  on  a  medium  composed  of  a  leucotoxic  serum  (pro- 
duced by  inoculating  calves  with  guinea-pig  leucocytes)  and  liver  tissue. 
Such  cultures,  he  claims,  contain  no  tuberculin.  To  the  sera  produced 
by  immunization  with  these  cultures  he  attributes  high  curative  powers. 

Bacilli  Closely  Related  to  the  Tubercle  Bacillus. — The  Bacillus  of 
Bovine  Tuberculosis. — Tuberculosis  of  cattle  (Perlsucht)  was  studied 
by  Koch  ^  in  connection  with  his  early  work  on  human  tuberculosis. 
Koch  did  not  fail  to  recognize  differences  between,  the  reactions  to  in- 
fection in  the  bovine  type  of  the  disease  and  that  of  man.  He  attrib- 
uted these,  however,  to  the  nature  of  the  infected  subject  rather  than 
to  any  differences  in  the  infecting  agents.  This  point  of  view  met 
with  little  authoritative  contradictibn,  until  Theobald  Smith, "^  in  1898, 
made  a  systematic  comparative  study  of  bacilli  isolated  from  man  and 
from  cattle  and  pointed  out  differences  between  the  two  types.  The 
opinion  of  Smith  was  fully  accepted  by  Koch  ^  in  1901. 

Since  that  time,  the  question,  because  of  its  great  importance  to 
prophylaxis,  has  been  the  subject  of  many  investigations,  most  of  them 

^Maragliano,  Berl.  klin,  Woch.,  1899;  Soc.  de  biol,  1897. 

2  Marmorek,  Berl.  klin.  Woch.,  1903,  p.  1108;   Med.  Klinik,  1906. 

^  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  11,  1882, 

*  Th.  Smith,  Jour.  Exp.  Med.,  Ill,  1898. 

6  i^oc/i,  Deut.  med.  Woch.,  1901.  '' 


THE  TUBERCLE  BACILLUS  499 

confirming  Smith's  original  work.  Morphologically,  Smith  ^  found  that 
the  bovine  bacilli  were  usually  shorter  than  those  of  the  human  type  and 
grew  less  luxuriantly  than  these  upon  artificial  media.  He  determined, 
furthermore,  that,  grown  upon  slightly  acid  glycerin  bouillon,  the  bovine 
bacillus  gradually  reduces  the  acidity  of  the  culture  medium  until  the 
reaction  reaches  neutrality  or  even  shght  alkalinity.  Fluctuations, 
after  this,  do  not  exceed  0.1  or  0.2  per  cent  on  either  side  of  neutrality. 
In  the  case  of  the  human  bacillus,  on  the  other  hand,  there  is  but  slight 
reduction  of  the  acidity  during  the  first  weeks  of  growth;  after  this 
acidity  increases  and,  though  subject  to  fluctuations,  never  reaches 
neutrality.  This  behavior  is  probably  due  to  action  exerted  upon  the 
glycerin,  since  on  ordinary  bouillon  no  such  differences  between  the  two 
varieties  can  be  noticed.  These  observations  of  Smith  were  confirmed 
by  Ravenel,^  Vagedes,^  and  others. 

The  cultural  differences  between  the  two  types  have  been  studied 
with  especial  care  by  Wolbach  and  Ernst,^  and  Kossel,  Weber,  and 
Heuss.^  All  of  these  observers  bear  out  Smith's  contention  that 
luxuriance  and  speed  of  growth  are  much  more  marked  in  the  human 
than  in  the  bovine  variety.  Marked  differences,  furthermore,  have  been 
shown  to  exist  in  the  pathogenic  qualities  of  these  bacilli  toward  various 
animal  species. 

Guinea-pigs  inoculated  with  the  bovine  type  ®  die  more  quickly  and 
show  more  extensive  lesions  than  those  infected  with  himian  bacilli. 
The  difference  in  the  pathogenicity  of  the  two  organisms  for  rabbits  is 
sufficiently  striking  to  be  of  diagnostic  value.  The  bovine  bacilli  usually 
kill  a  rabbit  within  two  to  five  weeks;  the  himaan  bacilli  produce  a  mild 
and  slow  disease,  lasting  often /for  six  months,  and  occasionally  fail  to 
kill  the  rabbits  at  all. 

The  practical  importance  of  distinguishing  between  the  two  types, 
of  course,  attaches  to  the  question  as  to  whether  the  bovine  and  the 
human  disease  are  mutually  intercommunicable.  Extensive  attempts  to 
infect  cattle  with  bacilli  of  the  human  type  have  been  made,^  for  the  most 
part  with  very  httle  or  no  success.     Infections  of  human  beings  with 

1  Th.  Smith,  Jour.  Exp.  Med.,  1905. 

2  Ravenel,  Lancet,  1901;  Univ.  Penn.  Med.  Bull.,  1902. 

3  Vagedes,  Zeit.  f.  Hyg.,  1898. 

^Wolbach  and  Ernst,  "Studies  from  the  Rockefeller  Inst.,"  11,  1904. 

^  Kossel,  Weber,  und  Hems,  Arb.  a.  d.  kais.  Gesundheitsamt,  1904  and  1905. 

^  Smith,  loc.  cit.,  and  Medical  News,  1902. 

7  Beck,  "Festsch.  R.  Koch,"  1902;  SmUh,  loc.  cit. 


500  PATHOGENIC  MICROORGANISMS 

bovine  bacilli,  however,  have  been  reported  and  proved  beyond  reason- 
able doubt,  by  Smith,i  Ravenel,^  Kossel,  Weber,  and  Heuss,^  Park  and 
Krumwiede,^  and  others.  Most  of  these  infections  have  been  in  children. 
It  is  likely,  therefore,  that  while  cattle  are  to  a  considerable  degree  im- 
mune against  the  bacillus  of  the  human  type,  human  beings  do  not 
enjoy  the  same  safeguard  in  respect  to  the  bovine  bacillus.  During  adult 
life,  the  danger  of  such  infection,  however,  is  far  less  than  it  is  during 
infancy  and  early  youth.    This  question  has  been  discussed  on  p.  487. 

The  Bacillus  of  Avian  Tuberculosis. — A  disease  resembling  in  many 
features  the  tuberculosis  of  man  is  not  uncommon  among  chickens, 
pigeons,  and  some  other  bird  species.  Koch  was  the  first  to  discover  in 
the  lesions  of  diseased  fowl  bacilli  much  resembling  Bacillus  tuberculosis. 
It  was  soon  shown,  however,  by  the  studies  of  Nocard  and  Roux,^ 
Mafucci,^  and  others,  that  the'bacillus  of  the  avian  disease  represented 
a  definitely  differentiable  species. 

Morphologically,  and  in  staining  characteristics,  the  bacillus  is 
almost  identical  with  that  of  the  human  disease.  In  culture,  however, 
growth  is  more  rapid  and  takes  place  at  a  temperature  of  41°  to  45°  C.^ 
(the  normal  temperature  of  birds),  while  the  human  type  is  unable  to 
thrive  at  a  temperature  above  40°. , 

Guinea-pigs,  very  susceptible  to  human  tuberculosis,  are  very 
refractory  to  infection  with  the  avian  type;  while,  on  the  other  hand, 
rabbits  which  are  resistant  to  the  human  type,  succumb  rapidly  to  in- 
fection with  avian  tuberculosis.^  Prolonged  cultivation  and  passage 
through  the  mammahan  body  is  said  to  cause  these  bacilli  to  approach 
more  or  less  closely  to  the  mammalian  type.  Conversely,  Nocard  ^ 
succeeded  in  rendering  mammalian  tubercle  bacilli  pathogenic  for  fowl 
by  keeping  them  in  the  peritoneal  cavities  of  hens  in  celloidin  sacs  for 
six  months. 

Recently  Koch  and  Rabinovitsch  ^^  have  isolated  from  the  spleen  of 

^  Smith,  Trans.  Assn.  Amer.  Phys.,  1903. 
2  Ravenel,  Univ.  Penn.  Med.  Bull.,  1902. 
^  Kossel,  Weber,  und  Heitss,  loc.  cit. 
*  Park  and  Krumwiede,  Jour.  Med.  Res.,  1910. 
^  Nocard  et  Roux,  Ann.  de  I'inst.  Pasteur,  1887. 
^  Mafucci,  Zeit.  f .  Hyg.,  xi. 
'  Mafucci,  loc.  cit. 

8  Straus  et  Gamaleia,  Arch,  de  m^d.  exp^r.,  1891;  Courmont  et  Dor,  Arch,  de  med. 
exp.,  1891. 

8  Nocard,  Ann.  de  I'inst.  Pasteur,  1898. 
^^Koch  und  Rabinovitsch,  Virch.  Arch.,  Beiheft  to  Bd.  190,  1907. 


THE   TUBERCLE   BACILLUS  501 

a  young  man  dead  of  tuberculosis,  a  microorganism  which,  culturally, 
morphologically,  and  in  its  pathogenic  action  upon  birds,  seemed  to 
belong  to  the  avian  type.  Lowenstein^  describes  a  similar  organism 
cultivated  from  a  human  case  which  seems  to  be  a  transitional  type. 
Observations  of  this  order  are,  however,  too  few  at  the  present  time  to 
be  used  as  the  basis  of  a  definite  opinion  as  to  the  relationship  between 
the  two  varieties. 

Tuberculosis  in  Cold-blooded  Animals. — The  bacillus  isolated  by 
Dubarre  and  Terre  ^  resembles  Bacillus  tuberculosis  in  morphology  and 
in  a  certain  degree  of  acid-fastness.  It  grows  at  low  temperatures, 
15°  to  30°  C.  It  is  non-pathogenic  for  animals,  but  kills  frogs  within  a 
month.  Except  for  the  acid-fastness  it  has  little  in  common  with 
Bacillus  tuberculosis. 

Similar  acid-fast  bacilli  have  been  isolated  from  other  cold-blooded 
animals  (carp,  frogs,  turtles,  snakes)  by  many  observers. 

There  have  been  many  attempts  to  show  a  close  relationship  between 
the  tubercle  bacilli  of  cold-blooded  and  those  of  warm-blooded  animals. 
Moeller,  Hansemann,  Friedmann,  Weber,  Ktister,  and  others  have 
given  this  subject  particular  attention  and  it  has  gained  especial  interest 
because  of  the  recent  notorious  claims  of  Friedmann  that  he  has  suc- 
ceeded in  obtaining,  from  turtles,  a  strain  of  acid-fast  bacilli  which  can 
be  successfully  used  in  actively  immunizing  human  beings.  In  1903 
Friedmann  ^  described  two  cases  of  spontaneous  infection  of  a  salt-water 
turtle  (Chelone  corticata)  with  acid-fast  bacilli,  presenting  lesions  in 
the  lungs  which  simulated  pulmonary  tuberculosis  in  the  human  being 
(cavity  formation  and  miliary  nodules) .  The  organisms  cultivated  from 
these  lesions  presented  much  similarity  to  those  of  the  human  type  and, 
according  to  Friedmann,^  unlike  other  acid-fast  bacilli  of  cold-blooded* 
animals,  could  be  grown  at  37.5°  C.  As  a  possible  human  origin  for  the 
turtle  infections  Friedmann  mentions  that  the  attendant  who  fed  these 
turtles  suffered  from  a  double  pulmonary  tuberculosis. 

Upon  inoculation  into  guinea-pigs  localized  lesions  only  were  pro- 
duced, and  dogs,  rats,  and  birds  were  inmiune.  The  implication  of 
Friedmann's  work  is  that  his  culture  represents  a  human  strain  attenu- 


^  Lowenstein,  quoted  from  Koch  and  Rabinovitsch,  loc.  cit. 
2  Dubarre  et  Terre,  Compt.  rend,  de  la  soc.  de  biol.,  1897. 
^Fnedmann,  D.  Med.  Woch.,  No.  2,  Jan.,   1903,  25. 

4  FHedmann,  D.  Med.  Woch.,  No.  26,  464,  1903,  and  Centralbl.  f .  Bakt.,  I,  xxxiv, 
1903,  also  Zeitschr.  f.  Tuberkulose,  iv,  Heft  5,  1903. 


502  PATHOGENIC   MICROORGANISMS 

ated  for  man  by  passage  through  the  turtle,  although,  as  far  as  we  are 
aware,  no  definite  statement  as  to  this  has  been  made. 

Summarizing  the  work  of  many  investigators  (Weber,  Taute, 
Ktister,  Allegri,  Eertarelli,  and  others)  Kiister  ^  makes  a  statement 
which  is,  in  essence,  as  follows:  In  the  carp,  in  snakes,  turtles, 
and  frogs  spontaneous  tuberculosis  may  occur.  The  organisms  which 
cause  these  diseases  are  specific  for  cold-blooded  animals,  similar  in 
many  respects  to  the  tubercle  bacillus  of  warm-blooded  animals,  but 
in  the  latter  do  not  produce  progressive  disease.  Human,  bovine,  and 
avian  tubercle  bacilli  inoculated  into  cold-blooded  animals  can  produce 
lesions  which  histologically  simulate  tuberculosis.  These  micro- 
organisms can  remain  a  year  in  cold-blooded  animals  without  losing 
their  pathogenicity  for  guinea-pigs.  Mutation  of  the  tubercle  bacillus 
of  warm-blooded  animals  into  cold-blooded  ones  has  not  been  proven. 

For  these  reasons  it  is  quite  impossible  to  exclude,  in  the  apparently 
positive  work  of  Friedmann  and  others,  the  isolation  of  a  true  "  cold- 
blooded" type  organism,  rather  than  a  mutation  form  originally  of 
the  warm-blooded  type.  What  Friedmann's  present  claims  in  this 
respect  are  for  his  culture  has  not  been  stated  as  far  as  we  know.  The 
possibility  of  a  positive  immunizing  value  of  organisms  isolated  from 
cold-blooded  animals  in  human  beings,  though  remote,  is  not  out  of  ques- 
tion. The  problem  is  so  serious  and  important,  and  the  experience  of 
many  workers  is,  so  far,  so  inconclusive  that  the  time  has  not  come  for 
commercial  exploitation  and  the  cruel  arousing  of  false  hopes.  The 
subject,  however,  deserves  carefully  controlled  further  investigations. 

Bacillus  of  Timothy. — Moeller  isolated  from  timothy-grass  and  from 
the  dust  in  haylofts  acid-fast  bacilli,  like  Bacillus  tuberculosis.  Thfey 
grow  rapidly  on  agar,  soon  showing  a  deep  red  or  dark  yellow  color.  ' 

Bacillus  butyricus  {Butter  Bacillus). — Slightly  acid-fast  bacilli  'i'e- 
sembling  Bacillus  tuberculosis  have  been  isolated  from  milk  and  butter 
by  Petri,2  Rabinovitsch,^  Korn,^  and  others. 

These  bacilli  are  easily  differentiated  from  Bacillus  tuberculosis  cul- 
turally.   They  are  slightly  pathogenic  for  guinea-pigs,  but  riot  for  man. 

Bacillus  smegmatis  and  the  bacillus  of  leprosy  will  be  discussed  in 
separate  sections.  The  differentiation  of  these  organisms  by  staining 
reactions  has  been  discussed  in  the  section  on  staining  methods. 

^  Kolle  und  Wassermann's  Handbuch,  2d  edition,  v,  767. 

2  Petri,  Arb.  a.  d.  kais.  Gesundheitsamt,  1897. 

3  Rabinovitsch,  Zeit.  f .  Hyg.,  1897. 
*  Korn,  Cent,  f .  Bakt.,  1899. 


CHAPTER  XXXIV 
THE  SMEGMA  BACILLUS  AND  THE   BACILLUS  OF   LEPROSY 

BACILLUS    SMEGMATIS 

In  1884,  Lustgarten  ^  announced  that  he  had  succeeded  in  demon- 
strating, in  a  number  of  syphilitic  lesions,  a  characteristic  bacillus, 
which  he  declared  to  be  the  etiological  factor  in  the  disease.  The  great 
importance  of  the  subject  of  Lustgarten's  communication  caused  nu- 
merous investigators  to  take  up  the  study  of  the  microorganisms  found 
upQn  the  genitals  of  normal  and  diseased  individuals.  As  a  result  of 
these  researches  the  presence  of  the  Lustgarten  bacilh  upon  the  genitals 
of  many  syphilitics  was  confirmed;  but  at  the  same  time  bacilli,  which 
in  all  essential  particulars  were  identical  with  them,  were  found  in  the 
secretions  about  the  genital  organs  and  anus  of  many  normal  persons. 
The  first  to  throw  doubt  upon  the  etiological  significance  of  Lustgarten's 
bacillus,  and  to  describe  in  detail  the  microorganism  now  recognized  as 
Bacillus  smegmatis,  were  Alvarez  and  Tavel.^  Similar  studies  were 
made  soon  afterward  by  Klemperer,^  Bitter,^  and  others. 

The  smegma  bacilli  are  now  known  to  occur  as  harmless  sapro- 
phytes in  the  preputial  secretions  of  the  male,  about  the  external  genital 
organs  of  the  female,  and  within  the  folds  of  thighs  and  buttocks.  They 
are  usually  found,  in  these  situations,  in  clumps  upon  the  .mucous  mem- 
brane, and  occasionally  in  the  superficial  layers  of  the  epithelium,  intra- 
and  extra-cellularly. 

Morphology. — The  smegma  bacilli  are  very  similar  to  tubercle  bacilli, 
but  show  greater  variations  in  size  and  appearance  than  do  the  latter. 
In  length  the  individuals  may  vary  from  two  to  seven  micra.  They 
are  usually  straight  or  slightly  curved,  but  according  to  Alvarez  and 
Tavel  may  show  great  polymorphism,  including  short  comma-like  forms, 
and  occasional  S-shaped  spiral  forms. 

1  Lustgarten,  Wien.  med.  Woch.,  47,  1884. 

2  Alvarez  et  Tavel,  Arch.  d.  physiol.  norm,  et  path.,  Oct.,  1885. 

3  Klemperer,  Deut.  med.  Woch.,  xi,  1885. 
*  Bitter  J  Virchow's  Arch.,  ciii. 

503 


504  PATHOGENIC   MICROORGANISMS 

They  are  not  easily  stained,  and  though  less  resistant  in  this  respect 
than  the  tubercle  bacillus,  they  yet  belong  distinctly  to  the  group  of 
acid-fast  bacilli.  Once  stained  by  the  stronger  dyes,  such  as  carbol- 
fuchsin  or  anilin-water-gentian-violet,  they  are  tenacious  of  the  dye, 
though  less  so  than  tubercle  bacilli. 

The  identification  of  the  smegma  bacillus  by  staining  methods  has 
become  of  great  practical  importance  since  Fraenkel,^  Miiller,^  and 
others  have  demonstrated  the  occasional  presence  of  acid-fast  bacilli, 
probably  of  the  smegma  group,  in  sputum,  and  in  secretions  from  the 
tonsillar  crypts  and  throat.  The  methods  of  differentiation  which  have 
been  found  most  practical  are  those  which  depend  upon  differences  in 
the  retention  of  stain  shown  by  these  bacilli.  While  it  may  be  stated 
as  a  general  rule  that  the  smegma  bacilli  are  more  easily  decolor- 
ized than  tubercle  bacilli,  it  is  nevertheless  important  that  a  con- 
trol, as  suggested  by  Wood,  be  made  with  known  tubercle  bacilli 
whenever  a  slide  of  suspected  smegma  bacilli  is  examined.  For 
the  actual  differentiation  an  excellent  method  is  that  of  Pappenheim, 
described  in  detail  in  the  section  on  Staining,  page  106.  This  method 
depends  upon  the  fact  that  prolonged  treatment  with  alcohol  and  rosolic 
acid  decolorizes  the  smegma  bacilli  but  not  the  tubercle  bacilli. 
Coles  ^  has  stated  that  smegma  bacilli  will  resist  Pappenheim's 
decolorizing  agent  for  four  hours  at  the  most,  while  tubercle  bacilli 
will  retain  the  stain,  in  spite  of  such  treatment,  for  as  long  as  twenty- 
four  hours. 

Although  minor  differences  between  the  smegma  bacillus  and  that 
of  Lustgarten  have  been  upheld  by  Doutrelepont  *  and  others,  never- 
theless, the  etiological  significance  of  Lustgarten's  bacillus  in  syphilis 
has  been  finally  discredited,  and,  if  not  identical  with  the  smegma 
bacillus,  it  at  least  belongs  to  the  same  group. 

The  smegma  bacilli  have  no  pathogenic  significance.  They  are 
found  upon  human  beings  as  harmless  saprophytes,  and  all  attempts  to 
infect  animals  have  so  far  been  unsuccessful.  They  are  cultivated 
with  great  difficulty,  first  cultivations  from  man  being  successful  only 
upon  the  richer  media  containing  human  serum  or  hydrocele  fluid. 
After  prolonged  cultivation  upon  artificial  media  they  may  be  kept 
alive  upon    glucose    agar    or    ascitic    agar.     Their    growth    is    slow; 

1  Fraenkel,  Berl.  klin.  Woch.,  1898. 

^Mailer,  Deut.  med.  Woch.,  1898. 

»  Coles,  Jour,  of  State  Med.,  1904. 

»  Doutrelepont,  quoted  from  Klemperer,  loc.  cit. 


BACILLUS  LEPR^  AND  LEPROSY  505 

and  the  colonies,  appearing  within  five  or  six  days  after  inoculation, 
are  yellowish  white,  corrugated,  and  not  unlike  tubercle-bacillus 
colonies. 

BACILLUS  LEPRAE  AND  LEPROSY 

The  bacillus  of  leprosy  was  first  seen  and  correctly  interpreted  as 
the  etiological  factor  in  the  disease  in  1879,  by  G.  Armauer  Hansen/ 
a  Norwegian  observer.  Hansen  found  the  bacilli  in  the  tissues  of  the 
nodular  lesions  of  patients,  lying  in  small  clumps,  intra-  and  extra- 
cellularly,  as  well  as  in  the  serum  oozing  from  the  tissue  during  its 
removal.  Hansen's  observation  was  the  fruit  of  over  six  years  of  careful 
study  and  as  to  his  priority  in  making  this  great  discovery,  there  can 
be  no  doubt.  Almost  simultaneously  with  his  publication,  however, 
Neisser  ^  published  similar  results,  obtained  by  him  during  a  brief  stay 
at  Bergen,  during  the  preceding  summer.  The  bacilli  described  by 
these  workers  are  now  recognized  as  being  unquestionably  the  cause 
of  the  various  forms  of  the  disease  known  as  leprosy. 

Morphology  and  Staining. — The  leprosy  bacillus  is  a  small  rod 
measuring  about  5  to  7 [x  in  length  and  has  a  close  morphological  re- 
semblance to  Bacillus  tuberculosis,  except  in  that  it  is  less  apt  to  display 
the  beaded  appearance  and  is  slightly  less  slender  than  the  latter.  It 
is  non-motile,  possesses  no  flagella,  and  forms  no  spores. 

Like  tubercle  bacilli,  furthermore,  the  leprosy  bacilli  belong  to  the 
class  of  so-called  acid-fast  bacteria,  being  stained  with  much  difficulty; 
but  when  once  stained  they  are  tenacious  of  the  color,  offering  con- 
siderable resistance  to  the  decolorizing  action  of  acids.  It  is  necessary 
for  differential  diagnosis,  however,  to  note  that  both  the  difficulty  of 
staining  and  the  resistance  to  decolorization  are  less  marked  in  the  case 
of  this  microorganism  than  in  the  case  of  Bacillus  tuberculosis.  It  was 
this  peculiar  behavior  to  stains  that  caused  the  delay  of  several  years  in 
Hansen's  publications,  since  he  failed  in  obtaining  good  morphological 
specimens  until  the  work  of  Koch  upon  bacterial  staining  had  supplied 
him  with  proper  methods.  The  bacillus  is  stained  most  easily  with 
anilin-water-gentian-violet  or  with  carbol-fuchsin  solution.  Stained  by 
Gram's  method,  it  is  not  decolorized  and  appears  a  deep  blue.  Differ- 
ential staining  by  the  Ziehl-Neelsen  method  shows  the  bacillus  stained 
red  unless  decolorization  by  means  of  the  acid  and  alcohol  are  prolonged 

1  Hansen,  Virch.  Arch.,  79,  1879. 

2  Neisser,  Breslauer  arztl.  Zeitschr.,  20,  1879, 


506  PATHOGENIC  MICROORGANISMS 

for  an  unusual  time.  A  differentiatian  from  tubercle  bacilli  by  virtue  of 
greater  ease  of  decolorization  is  of  value  only  in  the  hands  of  those 
having  much  experience  with  these  bacilli,  and  follows  no  regular  laws 
of  acid-strengths  or  time  of  application  which  can  be  generally  applied 
by  the  inexperienced.  In  tissues,  the  bacilli  are  easily  stained  by  the 
methods  used  for  staining  tubercle  bacilli.  The  sections  are*  left  in  the 
Ziehl  carbol-fuchsin  solution  either  from  two  to  twelve  hours  at  incu- 
bator temperature  or  for  twenty-four  hours  at  room  temperature. 
Subsequent  treatment  is  that  employed  in  the  case  of  tuberculous  tissue 
sections  (see  p.  112). 

Cultivation. — Cultivation  of  the  leprosy  bacillus  has  not  met  with 
success.  Hansen  and  others  who  have  approached  the  problem  with 
a  thorough  knowledge  of  the  microorganism,  combined  with  a  com- 
petent bacteriological  training,  have  failed  in  all  their  attempts. 
Numerous  positive  results  reported  by  observers  have  always  lacked 
adequate  confirmation.  Recently,  Rost,^  of  the  British  Army  Medical 
Corps,  has  claimed  success  in  cultivation  of  leprosy  bacilli  upon  salt-free 
bouillon,  his  point  of  departure  being  the  previous  observation  that 
salt-free  media  favored  the  growth  of  tubercle  bacilli.  His  results  have 
not  been  confirmed. 

In  1909  Clegg^  succeeded  in  growing  an  acid-fast  bacillus  from 
leprous  tissue,  obtaining  his  results  by  inoculating  leprous  material 
upon  agar  plates  upon  which  ameba  coli  had  been  grown  in  symbiosis 
with  other  bacteria.  On  such  plates  the  acid-fast  bacilli  multiplied, 
and,  subsequently,  pure  cultures  were  obtained  by  heating  the  cultures 
to  60°  C,  which  destroyed  the  ameba  coli  and  other  bacteria.  These 
results  were  confirmed  by  other,  workers  and,  soon  after  that,  Duval  ^ 
not  only  succeeded  in  repeating  Clegg's  experiments,  but  obtained  cul- 
tures of  an  acid-fast  bacillus  directly  from  leprous  lesions  without  the 
aid  of  ameba.  He  first  observed  that  the  leprosy  organism  would  multi- 
ply around  a  transplanted  piece  of  leprous  tissue  upon  ordinary  blood 
agar  tubes  upon  which  influenza  bacilli  and  meningococci  were  grown. 
He  concluded  that  such  growth  depended  upon  chemical  changes  in 
the  media  and  believed  the  formation  of  amino-acids  essential  for  the 
initial  growth.  The  method  he  subsequently  described  depended  upon 
supplying  these  substances  either  by  adding  tryptophan  to  nutrient 
agar  or  by  pouring  egg  albumen  and  human  blood  serum  in  Petri  dishes, 

1  Rost,  Brit.  Med.  Jour.,  1,  1905. 

2  Clegg,  Philippine  Jour,  of  Sc,  iv,  1909. 

3  Duval,  Jour.  Exp.  Med.,  xii,  1910,  and  ibid.,  15,  1912. 


BACILLUS  LEPR^  AND  LEPROSY  507 

inspissating,  at  70°  C,  for  three  hours  and,  after  inoculating  with 
leprous  tissue,  adding  a  1  per  cent  solution  of  trypsin.  Indirectly  the 
same  result  was  obtained  by  employing  culture  media  containing  albu- 
minous substances  and  inoculating  with  bacteria  capable  of  producing 
amino-acids  from  the  medium.  After  leprosy  bacilli  had  been  grown 
on  this  medium  for  several  generations,  they  could  easily  be  cultivated 
on  agar  slants  without  special  additions  or  preliminary  treatment. 

In  spite  of  extensive  work  upon  this  very  important  problem 
opinions  are  still  divided  as  to  the  specific  nature  of  the  organisms  cul- 
tivated by  Clegg  and  by  Duval.  Animal  experiments  with  these  cultures 
have  remained  inconclusive.  The  cultures  after  prolonged  preservation 
upon  artificial  media  grow  heavily,  often  lose  their  acid-fast  charac- 
teristics, develop  into  streptothrix-hke  or  diphtheroid  forms  and  become 
markedly  chromogenic,  all  these  characteristics  suggesting  saprophytism. 

In  a  recent  communication,  Duval  and  Wellman  ^  state  their  opinion 
as  follows :  From  29  cases  of  leprosy,  22  successive  cultivations  of  acid- 
fast  bacilli  were  made;  in  14  of  them  a  chromogenic  organism,  similar 
to  that  of  Clegg,  was  found.  This  grows  either  as  a  non-acid-f ast  strep- 
tothrix  in  subsequent  cultivations  or  as  non-acid-fast  diphtheroid  forms. 
From  eight  cases  an  organism  distinctly  different  from  the  former  t^s 
cultivated  which  grows  only  on  specific  media  and  by  serological  tests 
seems  to  give  reaction  which  differentiates  it  from  Clegg's  organism.  Du- 
val believes  that  there  is  no  reason  to  assume  specific  etiological  relation- 
ship for  the  first  organism  mentioned.  In  the  case  of  the  second,  he 
admits  that  not  sufficient  proof  has  been  brought,  but  states  his  belief 
that  its  etiological  significance  is  probable. 

Pathogenicity. — Innumerable  attempts  to  transmit  leprosy  to  ani- 
mals by  inoculation  have  been  unsuccessful.  Nicolle,^  however,  has 
recently  claimed  'successful  experiments  upon  monkeys  (macacus)  in 
whom  inoculation  with  tissue  from  infected  human  beings  was  followed, 
in  sixty-two  days,  by  the  development  of  a  small  nodule  at  the  site  of 
inoculation,  in  which,  upon  excision,  leprosy  bacilli  were  found.  In 
most  cases,  however,  inoculation  has  given  rise  merely  to  a  transient 
inflammatory  reaction. 

Among  human  beings,  leprosy  has  been  a  widely  spread  disease  since 
the  beginning  of  history,  and  much  evidence  is  found  in  ancient  lit- 
erature which  testifies  to  a  wide  distribution  of  the  disease  long  before 
the  Christian  era  and  throughout  the  Middle  Ages.    At  the  present  day, 

1  Duval  and  Wellman,  Jour,  of  Inf.  Dis.,  xi,  1912. 

2  Nicolle,  Sem.  medicale,  10,  1905. 


508  PATHOGENIC    MICROORGANISMS 

leprosy  is  most  common  in  the  eastern  countries,  especially  in  India  and 
China.  In  Europe  the  disease  is  found  in  Norway,  in  Russia,  and  in 
Iceland.  In  other  European  countries,  while  the  disease  occurs,  it  is 
not  at  all  common.  In  the  United  States,  there  are,  according  to  Osier, 
three  important  centers  of  leprosy  situated  in  Louisiana,  in  California, 
and  among  the  Norwegian  settlers  in  Minnesota.  The  disease  is  also 
present  in  several  provinces  of  Canada.  In  all  countries  in  which 
segregation  of  lepers  is  rigidly  practiced,  the  disease  is  diminishing.  In 
Norway,  according  to  Hansen,  proper  sanitary  measures  have  reduced 
the  number  of  lepers  from  2,870  in  1856,  to  577  in  1900. 

Clinically,  the  disease  appears  in  two  chief  varieties,  tubercular 
leprosy  and  the  so-called  anesthetic  leprosy.  In  the  former  variety, 
hard  nodular  swellings  appear,  usually  in  the  face,  but  often  on  other 
parts  of  the  body  as  well.  These  lead  to  frightful  disfigurement  and 
are  accompanied  by  a  falling-out  of  hair  and  a  loss  of  sensation  in  the 
affected  areas.  In  the  anesthetic  form,  there  is  usually  at  first  pain  in 
definite  areas  of  the  extremities  and  the  trunk,  which  is  soon  followed 
by  the  formation  of  flat  or  slightly  raised  pigmented  areas,  within  which 
there  is  absolute  anesthesia  with,  later,  atrophy  and  often  secondary 
necrosis  in  the  atrophied  parts.  The  disease  is  usually  chronic  in  its 
course. 

The  bacilli  are  found  in  large  numbers  in  the  cutaneous  lesions.  In 
the  knobs  of  the  nodular  variety,  they  lie  in  clumps  between  the  con- 
nective-tissue cells  and  within  the  large  spheroidal  cells  which  make  up 
the  nodules.  They  are  found,  also,  in  advanced  cases,  in  the  liver  and 
in  the  spleen,  lying  within  the  cells,  and,  to  a  slighter  extent,  in  the 
intercellular  spaces.  They  have  also  been  found  within  the  kidneys, 
the  endothehum  of  the  blood-vessels,  and  in  the  testicles.^  In  the  blood, 
the  bacilli  have  frequently  been  demonstrated,  especially  during  the 
febrile  attacks  which  occur  during  the  disease.  Westphal  and  Uhlen- 
hut  ^  have  found  the  bacilli  within  the  central  nervous  system, 
and  these  observers,  as  well  as  others,  have  found  them  Ijdng 
within  the  substance  of  the  peripheral  nerves,  thus  explaining  the 
anesthesia.  A  fact  of  enormous  importance  to  the  question  of 
transmission  is  the  observation  made  by  various  observers,  more 
especially  by  Sticker,  that  the  bacilli  are  found  with  great  regu- 
larity in  considerable  numbers  in  the  nasal  secretions  of  persons 
suffering  from  the  disease.     Sticker  is  inclined  to  regard  the  nose 

1  Sticker,  Miinch.  med.  Woch.,  39,  1897. 

2  Westphal  uud  UhUnhut,  Klin.  Jahrb.,  190X. 


BACILLUS  LEPR.E  AND  LEPROSY  509 

as  the  primary  path  of  infection.  Whether  or  not  this  be  true  can 
not,  at  present,  be  decided.  As  a  source  of  infection,  however,  the 
nasal  mucus  and,  secondarily,  the  saliva,  are  certainly  the  vehicles 
by  which  large  numbers  of  the  bacilli  leave  the  infected  patient,  and, 
therefore,  tend  to  spread  the  disease. 

The  contagiousness  of  leprosy  is  far  less  than  is  that  of  most  other 
bacterial  diseases.  Physicians  and  others  who  come  into  direct  contact 
with  large  numbers  of  leprous  patients,  observing  at  the  same  time  the 
ordinary  precautions  of  cleanliness,  rarely  contract  the  disease.  On 
the  other  hand,  intimate  contact  with  lepers  without  such  precautions 
is  the  only  possible  means  of  transmission.  The  demonstration  of 
leprosy  bacilli  in  dust,  soil,  etc.,  must  always  be  looked  upon  with  sus- 
picion, since,  apart  from  actual  human  inoculation,  there  is  no  method 
of  positively  differentiating  the  baciUi  from  similar  acid-fast  organisms. 
Instances  of  transmission  by  contact  are  on  record,  not  the  least  famous 
of  which  is  the  case  of  Father  Damien,  who  contracted  the  disease  while 
taking  care  of  the  lepers  upon  the  island  of  Molokai.  Hansen  states 
that  in  his  knowledge  no  case  of  leprosy  can  be  found  in  which  careful 
examination  of  the  past  history  will  not  reveal  direct  contact  with  a 
previous  case.  Direct  inoculation  of  the  human  being  with  material 
from  a  leprous  patient  has  been  successfully  carried  out  by  Arning,^ 
upon  a  Hawaiian  criminal.  In  this  case  a  piece  of  a  leprous  nodule 
was  planted  into  the  subcutaneous  tissue  of  the  left  arm.  One  month 
after  the  inoculation,  pain  appeared  in  the  arm  and  shoulder,  and  four 
and  a  half  months  later  a  typical  leprosy  nodule  was  formed.  Four 
years  after  the  inoculation,  the  patient  was  a  typical  leper. 

Although  our  inabihty  to  cultivate  the  leprosy  bacillus,  and  the  lack 
of  success  attending  animal  inoculation,  have  made  it  impossible  to  study 
more  closely  the  toxic  action  of  this  microorganism,  there  is,  neverthe- 
less, some  evidence  which  points  toward  the  production  of  a  poisonous 
substance  of  some  kind  by  the  bacillus.  Rost,^  who  claims  to  have 
cultivated  the  bacillus,  manufactured  from  his  cultures,  by  the  technique 
for  the  production  of  ^'Old  Tuberculin,"  a  substance  which  he  (Jailed 
"leprolin,"  and  which  he  employed  therapeutically  in  the  same  manner 
in  which  tuberculin  is  employed  in  tuberculosis.  As  stated  before,  the 
results  of  Rost  still  lack  confirmation.  Of  far  greater  importance,  both 
in  demonstrating  the  probability  of  the  existence  of  a  definite  toxin  as 
well  as  in  indicating  the  close  relationship  between  the  leprosy  bacillus 

1  Aming,  Vers.  d.  Naturfor.  u.  Aerzte,  1886.  ^  Rost,  loc.  cit. 


510  PATHOGENIC   MICROORGANISMS 

and  the  Bacillus  tuberculosis,  are  the  investigations  upon  the  action 
of  tuberculin  upon  leprous  patients.  When  tuberculin  is  adminis- 
tered to  lepers,  a  febrile  reaction  occurs  usually  twenty-four  or  more 
hours  after  the  administration.  The  fever  differs  from  that  produced 
by  the  use  of  the  same  substance  in  tuberculous  patients  in  that  it  is  of 
late  occurrence  and  lasts  considerably  longer.  At  the  same  time,  there 
may  be  marked  redness  and  tenderness  of  the  nodules.  In  isolated 
cases,  Babes  ^  has  noticed  alarmingly  high  and  prolonged  fever  together 
with  systemic  symptoms  such  as  nausea,  headache,  and  even  uncon- 
sciousness, following  the  injection  of  tuberculin.  The  same  writer 
claims  to  have  extracted  from  the  organs  of  lepers,  which  contained 
enormous  numbers  of  bacilli,  substances  which  showed  an  action  similar 
to  that  of  the  tuberculin. 

RAT    LEPROSY 

Stefansky  ^  first  observed  this  disease  among  rats  in  Odessa,  and 
since  then  it  has  been  observed  in  Berlin  (Rabinovitsch  ^),  in  London 
(Dean^),  in  New  South  Wales  (Tidswell  ^),  and  in  San  Francisco 
(Wherry  ^  and  McCoy  ^) .  The  disease  occurs  spontaneously  among 
house  rats  and  is  characterized  by  subcutaneous  induration,  swelling  of 
lymph  nodes,  with,  later,  falling  out  of  the  hair,  emaciation,  and  some- 
times ulceration.  Its  course  is  protracted  and  rats  may  live  with  it 
for  six  months  or  a  year.  When  a  rat  suffering  from  this  disease  is  dis- 
sected there  is  usually  found,  under  the  skin  of  the  abdomen  or  flank,  a 
thickened  area  which  has  the  appearance  of  adipose  tissue  except  that 
it  is  more  nodular  and  gray  and  less  shiny  than  fat.  It  is  so  like  fat, 
however,  that  it  is  often  possible  to  overlook  it  as  evidence  of  disease 
by  one  unfamiliar  with  the  condition.  In  this  area  acid-fast  bacilli 
looking  like  the  Bacillus  leprae  are  found  in  large  numbers.  These 
bacilli  are  also  found  in  the  lymph  nodes  and  sometimes  in  small  nodules 
which  appear  in  the  liver  and  lung. 

1  Babes,  in  KoUe  und  Wassermann,  "Handbuch,"  etc.,  Erst.  Erganz.  Bd.,  1907. 

^Stefansky,  Centralbl.  f.  Bakt.,  xxxiii,  481. 

'  Rabinovitsch,  Centralbl.  f.  Bakt.,  xxxiii,  577. 

^  Dean,  Centralbl.  f.  Bakt.,  xxxiv,  222;  Jour.  Hyg.,  xcix. 

^  Tidswell,  cited  by  Brinkerhoff  in  "The  Rat  and  Its  Relation  to  Public  Health," 
Treas.  Dept.,  Wash.,  1910. 

6  Wherry,  J.  A.  M.  A.,  June  6,  1908,  p.  1903;  Jour.  Inf.  Dis.,  dvii,  Rep.  U.  S. 
P.  H.,  and  M.  H.  S.,  xxiii,  1841. 

""  McCoy,  Rep.  U.  S.  P.  H.  and  M.  H.  S.,  xxiii,  981;  Abstr.  in  J.  A.  M.  A., 
Aug.  22,  1908,  690. 


RAT   LEPROSY  511 

The  disease  can  be  transmitted  experimentally  from  rat  to  rat  and 
probably  is  transmitted  naturally  from  rat  to  rat  by  the  agency  of 
fleas  (Wherry,  McCoy).  Although  clinically  not  exactly  like  human 
leprosy  the  condition  is  sufficiently  like  it  to  arouse  much  hygienic 
interest.  The  distribution  of  the  disease  in  various  parts  of  the  world 
does  not  correspond  with  the  distribution  of  leprosy.  A  peculiar  feature 
of  its  distribution  is  the  fact  that  in  San  Francisco,  as  the  writer  was 
told  by  McCoy,  almost  all  the  rats  that  suffered  from  this  disease  came 
from  the  district  in  which  the  retail  meat  business  is  located,  known  as 
"Butchertown."  The  organisms  were  made  to  multiply  in  vitro  by 
Zinsser  and  Cary  in  plasma  preparations  of  growing  rat  spleen.  Chapin 
has  succeeded  in  cultivating  them  by  a  method  analogous  to  the  trypsin- 
egg  albmnen  method  employed  by  Duval.  In  the  experiments  of  Zinsser 
and  Cary  it  was  found  that  although  the  organisms  may  retain  their 
acid-fast  characteristics  for  many  weeks  within  leucocytes  they  degen- 
erate rapidly  within  the  spleen  cells,  a  fact  which  seems  to  have  some 
bearing  on  the  mechanism  of  resistance  possessed  by  the  body  against 
acid-fast  organisms. 


34 


CHAPTER  XXXV 

BACILLUS    DIPHTHERIiE,    BACILLUS    HOFFMANNI,   AND    BACILLUS 

XEROSIS 

BACILLUS  DIPHTHERIiE 

Since  1821,  when  Bretonneau  of  Tours  published  his  observa- 
tions, diphtheria  has  been  an  accurately  recognized  clinical  entity. 
Our  knowledge  of  the  disease  in  the  sense  of  modern  bacteriology, 
however,  begins  with  the  first  description  of  Bacillus  diphtheriae  by 
Klebs  in  1883.  Klebs  ^  had  observed  in  the  pseudomembranes  from 
diphtheritic  throats,  bacilli  which  in  the  light  of  more  recent  knowledge 
we  can  hardly  fail  to  recognize  as  the  true  diphtheria  organism.  His 
v/ork,  however,  was  purely  morphological  and,  therefore,  inconclusive. 
One  year  after  this  announcement,  Loeffler  ^  isolated  and  cultivated  an 
organism  which  corresponded  in  its  morphological  characters  to  the  one 
described  by  Klebs.  He  obtained  it  from  thirteen  clinically  unques- 
tioned cases  of  diphtheria,  and,  by  inoculating  it  upon  the  injured  mucous 
surfaces  of  animals,  succeeded  in  producing  lesions  which  resembled 
closely  the  false  membranes  of  the  human  disease.  His  failure  to  find 
the  bacillus  in  all  the  cases  he  examined,  his  finding  it,  in  one  instahce, 
in  a  normal  throat,  and  his  inability  to  explain  to  his  own  satisfaction 
some  of  the  systemic  manifestations  of  the  infection  which  we  now 
know  to  be  due  to  the  toxin,  caused  him  to  frame  his  conclusions  in 
a  tone  of  the  utmost  conservatism.  The  second  and  third  publications 
of  Loeffler,^  however,  and  the  inquiry  into  the  nature  of  the  toxins 
produced  by  the  bacillus,  published  in  1888  by  Roux  and  Yersin/ 
eliminated  all  remaining  doubt  as  to  the  etiological  relationship  existing 
between  this  organism  and  the  disease. 

Innumerable  observations,  both  clinical  and  bacteriological,  by 
other  workers,  have,  since  that  time,  confirmed  the  early  investigations, 


1  Klebs,  Verb.  d.  2.  Kongr.  f.  inn.  Medizin,  Wiesbaden,  1883, 

2  Loeffler,  Mittheil.  a.  d.  kais.  Gesundheitsamt,  1884. 
^Loeffler,  Cent.  f.  Bakt.,  1887  and  1890. 

*  Roux  and  Yersin,  Ann.  de  I'inst.  Pasteur,  1888  and  1889. 
512 


BACILLUS  DIPIITHERIiE 


513 


and  it  is  to-day  a  scientific  necessity  to  find  the  bacillus  of  Klebs  and 
Loeffler  in  the  lesion  before  a  diagnosis  of  '' diphtheria '^  can  properly 
be  made. 

Morphology  and  Staining. — While  Bacillus  diphtherise  presents 
certain  characteristic  appearances  which  facilitate  its  recognition,  it  is, 
at  the  same  time,  subject  to  a  nimiber  of  morphological  variations  with 


i«i 


i^ 


«?  A"^?^ 


?irit>i JJ*-*:  *>  %>'l\yC  !  ^-  It  V  « 


^ 


/^i 


Fig.  105. — Bacillus  diphtheria. 


all  of  which  it  is  important  to  be  familiar.  These  variations  are,  to  a 
limited  extent,  dependent  upon  the  age  of  the  culture  and  upon  the 
constitution  of  the  medium  on  which  it  has  been  grown.  These 
factors,  however,  do  not  control  the  appearance  of  the  organism  with 
any  degree  of  regularity,  and  any  or  all  of  its  various  forms  may  occur 
in  one  and  the  same  culture.  It  is  likely  that  these  different  appear- 
ances represent  stages  in  the  growth  and  degeneration  of  the  indi- 
vidual bacilli,  but  there  does  not  seem  to  be  any  just  reason  for 
believing  that,  as  several  observers  have  stated,  there  is  definite  correla- 
tion between  its  microscopic  form  and  its  biological  characteristics,  such 
as  virulence,  toxicity,  etc. 


514  PATHOGENIC  MICROORGANISMS 

The  bacilli  are  slender,  straight,  or  slightly  curved  rods.  In  length 
they  vary  from  1.2  micra  to  6.4  micra,  in  breadth  from  0.3  to  1.1.  As 
seen  most  frequently  when  taken  from  the  throat  they  are  about  4  to 
5  micra  in  length.  They  are  rarely  of  uniform  thickness  throughout 
their  length,  showing  club-shaped  thickening  at  one  or  both  ends. 
Occasionally  they  may  be  thickest  at  the  center  and  taper  toward  the 
extremities.  When  thickened  at  one  end  only,  a  slender  wedge-shape 
results.  Such  forms  are  usually  straight,  of  smaller  size  than  their 
neighbors,  and  are  more  often  stained  with  great  uniformity.  These 
are  spoken  of  by  Beck  ^  as  the  "ground  type,"  and  assumed,  for  in- 
sufficient reasons,  to  be  the  young  individuals.  Branched  forms 
have  been  described  by  some  investigators.  They  are  rare  and 
probably  to  be  regarded  as  abnormal  or  involution  forms  due  to  un- 
favorable environment. 

The  organisms  stain  with  the  aqueous  anilin  dyes.  A  characteristic 
irregularity  of  staining  which  is  of  great  aid  in  diagnosis  is  best  obtained 
with  Loeffler's  "  alkaline  methylene-blue."  (For  preparation  see  section 
on  Staining,  p.  96.)  Stained  with  this  solution  for  five  to  ten  minutes 
many  of  the  bacilli  appear  traversed  by  unstained  transverse  bands 
which  give  them  a  striped  or  beaded  appearance.  The  longer  indi- 
viduals often  have  a  strong  resemblance  to  short  chains  of  strepto- 
cocci. Others  may  appear  unevenly  granular.  In  cultures  which 
are  about  eighteen  hours  old,  ma^y  of  the  bacilli  may  show  deeply 
stained  oval  bodies  situated  most  frequently  at  the  ends.  These  are 
the  so-called  " polar '^  or  "Babes-Ernst"  bodies.^  Special  stains  have 
been  devised  for  the  demonstration  of  these  appearances.  One  of  these 
was  originated  by  Neisser,^  who  claims  for  it  differential  value  in 
distinguishing  these  organisms  from  pseudodiphtheria  and  xerosis 
baciUi. 

His  method  requires  two  solutions: 

1.  Methylene  blue  (Griibler) 1  gram. 

Alcohol,  96  per  cent 20    c.c. 

Glacial  acetic  acid 50     " 

Water 950    " 

2.  Bismarck  brown 2  grams. 

Water 1,000   c.c. 

1  Beck,  in  KoUe  imd  Wassermann,  ii,  p.  773. 
a  Babes,  Zeit.  f.  Hyg.,  Bd.  v,  1889.      . 
»  Neisser,  Zeit.  f.  Hyg.,  xxiv,  1897. 


BACILLUS  DIPHTHERIA  515 

The  cover-slip  preparation,  after  having  been  fixed,  is  stained  with  so- 
lution No.  1  for  one  to  three  seconds.  It  is  then  washed  in  water  and 
immersed  for  from  three  to  five  seconds  in  solution  No.  2.  With  this 
stain  the  bodies  of  the  bacilli  appear  brown,  the  polar  granules  blue. 

Another  method  which  has  been  extensively  used  is  that  of  Roux. 
The  solutions  required  for  this  are: 

1 .  Dahlia  violet 1  gram. 

Alcohol,  90  per  cent 10  c.c. 

Aq.  dest ad  100    " 

2.  Methyl  green ' 1  gram. 

Alcohol,  90  per  cent 10  c.c. 

Aq.  dest ad  100    " 

The  two  solutions  are  mixed,  one  part  of  1  being  added  to  three  parts 
of  2.  Preparations  are  stained  in  this  mixture  for  two  minutes.  The 
polar  bodies  appear  a  dark  violet.  Other  methods  for  the  staining  of 
polar  bodies  have  been  recommended. 

The  significance  of  the  polar  bodies  is  not  well  understood.  Their 
discoverer,  Ernst,  regarded  them  as  bodies  analogous  to  the  spores  of 
other  organisms.  The  ease  with  which  they  are  stained,  however,  and 
the  low  temperatures  to  which  the  bacteria  succumb  make  this  appear 
very  unlikely.  A  more  probable  interpretation  seems  to  be  that  of 
Escherich  ^  who  regards  them  as  chromatic  granules. 

Stained  by  Gram's  method,  the  diphtheria  bacilli  retain  the  gentian- 
violet. 

In  stained  smears  from  the  throat  or  from  cultures  a  characteristic 
grouping  of  the  bacilli  has  been  observed.  They  lie  usually  in  small 
clusters,  four  or  five  together,  parallel  to  each  other,  or  at  sharp  angles. 
Two  organisms  may  often  be  seen  attached  to  each  other  by  their  cor- 
responding ends  while  their  bodies  diverge  to  form  a  ''V"  or  "Y"' shape. 

Biological  Characteristics. — The  diphtheria  bacillus  is  a  non-motile, 
non-flagellated,  non-spore-forming  aerobe.  Its  preference  for  oxygen 
is  marked,  but  it  will  grow  in  anaerobic  environment  in  the  presence  of 
suitable  carbohydrates.  It  does  not  liquefy  gelatin.  The  bacillus  grows 
at  temperatures  varying  between  19°  C.  and  42°  C,  the  most  favorable 
temperature  for  its  development  being  37.5°  C.  Temperatures  above 
37.5°,  while  not  entirely  stopping  its  growth,  impede  the  development 
of  its  toxin. 

Resistance. — The  thermal  death  point  of  this  organism  is  58°  C.  for 
•  ten  minutes,  according  to  Welch  and  Abbott.    Boiling  kills  it  in  about 

^Escherich,  "Aetologie,  etc.,  d.  Diphth.,"  Wien,  1894. 


"" 


516  PATHOGENIC  MICROORGANISMS 


one  minute.  Low  temperatures,  and  even  freezing,  are  well  borne. 
Desiccation  and  exposure  to  light  are  not  so  fatal  to  this  organism  as 
to  most  of  the  other  pathogenic  bacteria.  Sternberg  ^  has  found  it 
alive  in  dried  bits  of  the  pseudomembrane  after  fourteen  weeks.  It  is 
easily  killed  by  chemical  disinfectants  in  the  strengths  customarily 
employed.  H2O2  seems  especially  efficacious  in  killing  the  organisms 
rapidly. 

Cultivation. — The  diphtheria  bacillus  grows  readily  on  most  of  the 
richer  laboratory  media.  It  will  grow  upon  media  made  of  meat  ex- 
tract, but  develops  more  luxuriantly  on  all  those  which  have  a  meat 
infusion  as  their  basis.  While  it  will  grow  upon  both  acid  and  alkaline 
media,  it  is  sensitive  to  the  extremes  of  both,  the  most  favorable  reaction 
for  its  development  being  probably  about  0.5  per  cent  alkalinity  ex- 
pressed in  terms  of  f  NaOH.  Animal  proteids  added  to  the  media, 
in  the  form  of  blood  serum,  ascitic  fluid,  or  even  whole  blood,  increase 
greatly  the  rapidity  and  richness  of  its  growth.  Horse  serum  is  sup- 
posed by  some  to  be  especially  favorable. ^ 

Loeffler's  Medium. — ^The  most  widely  used  medium  for  the  cultiva- 
tion of  this  bacillus  is  the  one  devised  by  Loeffler.    This  consists  of: 

Beef  blood  serum 3  parts 

One  per  cent  glucose  meat-infusion  bouillon 1  part 

The  mixture  is  coagulated  at  70°  C.  in  slanted  tubes  and  sterilized  at 
low  temperatures  by  the  fractional  method.  Upon  this  medium  the 
diphtheria  bacillus  in  twelve  to  twenty-four  hours  develops  minute, 
grayish-white,  glistening  colonies.  These  enlarge  rapidly,  soon  out- 
stripping the  usually  accompanying  streptococci.  The  medium  seems 
to  possess  almost  selective  powers  for  the  bacillus  and,  for  this  reason, 
it  is  especially  valuable  for  diagnostic  purposes. 

Meat-Infusion  Agar. — Upon  slightly  alkaline  meat-infusion  agar  the 
bacillus  develops  readily,  though  less  so  than  on  Loeffler's  serum.  Or- 
ganisms which  have  been  on  artificial  media  for  one  or  more  genera- 
tions may  grow  with  speed  and  luxuriance  upon  this  medium.  When 
planted  directly  from  the  human  or  animal  body  upon  agar,  however, 
growth  may  occasionally  be  slow  and  extremely  delicate.  Colonies  on 
agar  appear  within  twenty-four  to  thirty-six  hours  as  small,  rather  trans- 
lucent, grayish  specks.  The  appearance  of  these  colonies  is  quite  char- 
acteristic and  easily  recognized  by  the  practiced  observer.    Surface  colo- 

1  Sternberg,  "Manual  Bac,"  p.  455.  2  Michel,  Cent.  f.  Bakt.,  1897. 


BACILLUS  DIPHTHERIiE  517 

nies  are  irregularly  round  or  oval,  showing  a  dark,  heaped-up,  nucleus- 
like center,  fringed  about  by  a  loose,  coarsely  granular  disk.  The  edges 
have  a  peculiarly  irregular,  torn  appearance  which  distinguishes  them 
readily  from  the  sharply  defined  streptococcus  colonies.  For  these 
reasons  agar  is  the  medium  most  commonly  used  for  purposes  of  iso- 
lation. 

The  addition  of  dextrose  1  per  cent,  nutrose  2  per  cent,  or  glycerin 
6  per  cent,  renders  agar  more  favorable  for  rapid  growth,  but  unfits  it 
for  the  preservation  of  cultures,  the  organism  dying  out  more  rapidly, 
probably  because  of  acid  formation. 

Meat-Infusion  Broth. — Upon  beef  or  veal  broth  the  diphtheria  bacil- 
lus grows  rapidly,  almost  invariably  forming  a  pellicle  upon  the  surface, 
— another  expression  of  its  desire  for  oxygen.  The  broth  remains  clear. 
Broth  tubes  with  such  growth,  therefore,  have  a  characteristic  appear- 
ance. 

Meat-infusion  gelatin  is  a  favorable  medium  for  the  Klebs-Loeffler 
bacillus,  but  growth  takes  place  slowly  because  of  the  low  temperature 
at  which  this  medium  must  be  kept.     Gelatin  is  not  fluidified. 

Milk  is  an  excellent  medium,  and  for  this  reason  may  even  occa- 
sionally be  a  vehicle  of  transmission.  There  is  no  coagulation  of  the 
milk. 

Upon  potato,  B.  diphtheriae  will  grow  only  after  neutralization  of  the 
acid.    It  is,  at  best,  however,  a  poor  nutrient  medium. 

Upon  the  various  pepton  solutions  the  bacillus  of  diphtheria  produces 
no  indol. 

Many  special  media  have  been  recommended  for  the  cultivation  of 
this  organism.  The  most  important  of  these  are  the  modification  of 
Loeffler's  serum  devised  by  Beck,^  the  horse-blood-fibrin  cake  used  by 
Escherich,  and  Wassermann's  ascitic-fluid-nutrose-agar,  called  by  him 
"Nasgar."  None  of  these  has  sufficient  advantages  over  the  simpler 
media,  however,  to  make  its  substitution  desirable. 

Isolation. — Cultures  are  taken  from  throats  upon  Loeffler's  blood 
serum.  These  are  permitted  to  grow  at  37.5°  C.  for  from  eighteen  to 
twenty-four  hours.  At  the  end  of  this  time  about  5  c.c.  of  bouillon  are 
poured  into  the  tubes  and  the  growth  is  gently  emulsified  in  the  broth 
with  a  platinum  loop.  Two  or  three  loopfuls  of  this  emulsion  are  then 
streaked  over  the  surface  of  glucose  agar,  serum  agar,  or  nutrose  agar. 
After  twenty-four  hours'  incubation  these  plates  show  characteristic 

.  1  M,  Beck,  KoUe  imd  Wasserjnann;  Brit.  Med.  Jour. 


518 


PATHOGENIC  MICROORGANISMS 


colonies  which  can  be  easily  fished  and  again  transferred  to  Loeffler 
tubes  or  any  other  suitable  medium. 

Diagnosis. — Cultures  from  suspected  throats  are  taken  on  Loeffler 's 
blood  serum  medium  and  incubated  at  37.5°  C.  for  12  to  18  hours.  At 
the  end  of  this  time  morphological  examination  by  staining  with  Loef- 
fler's  alkaline  methylene  blue  and  by  some  polar  body  stain  like  that 

of  Neisser  is  carried  out.  Occa- 
sionally direct  smears  from  the 
throat  may  show  the  bacilli,  but  it 
is  rarely  possible  to  make  a  satis- 
factory diagnosis  in  this  way. 

Williams  has  pointed  out  that  in 
throat  cultures  in  which  the  diph- 
theria bacilli  are  few  in  number 
it  is  of  advantage  to  inoculate  a 
tube  of  ascitic  broth  with  the 
mixed  culture.  The  diphtheria  ba- 
cilli will  appear  in  eighteen  to 
twenty-four  hours  as  a  pellicle  on 
the  surface.  A  portion  of  this 
pellicle  may  then  be  plated  on 
ascitic  agar  and  isolated  in  pure 
culture  from  the  colonies. 
Pathogenicity. — Bacillus  diphtherias  causes  a  more  or  less  specific 
local  reaction  in  mucous  membranes,  which  results  in  the  formation  of 
the  so-called  *  *  pseudo-membranes. ' '  When  these  are  characteristically 
present,  infection  with  this  bacillus  should  always  be  suspected.  The 
consequent  disease  depends,  in  part,  upon  the  mechanical  disturbance 
caused  by  these  false  membranes  and,  in  part,  upon  the  systemic  poi- 
soning with  the  toxin  which  the  bacilli  produce.  Although  the  diph- 
theria bacillus  has  been  found  after  death  in  the  spleen  and  liver,  we 
have  no  data  which  would  justify  the  assumption  that  a  true  diph- 
theria-septicemia  may  occur  during  life.  It  is  probable  that  in  those 
cases  which  Baginsky  ^  has  called  the  septicemic  form  of  diphtheria. 
Bacillus  diphtherias  has  merely  opened  a  path  by  which  accompanying 
streptococci  have  gained  access  to  the  lymphatics  and  the  blood  stream. 
The  most  frequent  sites  of  diphtheritic  inflammation  are  the  mucous 
membranes  of  the  throat,  larynx,  and  nose.     They  have  also  been 


Fig.  106. — Colonies  of  Bacillus 

DIPHTHERIA  ON  GlYCERIN  AgAR. 


^Baginsky^  "Lehrbuch  d,  Kinderkrankheiten/ 


BACILLUS  DIPHTHERIA  619 

found  in  the  car,  upon  the  mucous  membrane  of  the  stomach  and  the 
valva,  and  upon  the  conjunctiva  and  the  skin.  According  to  Loeffler, 
Strelitz/  and  others,  the  bacillus  may,  by  extension  from  the  larynx, 
give  rise  to  a  true  diphtheritic  broncho-pneumonia. 

For  the  usual  laboratory  animals  the  diphtheria  bacillus  is  very 
pathogenic.  Dogs,  cats,  fowl,  rabbits,  and  guinea-pigs  are  susceptible. 
Rats  and  mice  are  resistant.  False  membranes,  analogous  to  those 
found  in  human  beings,  have  been  produced  in  many  animals,  but 
only  when  inoculation  had  been  preceded  by  mechanical  injury  of  the 
mucosa.  Small  quantities  (0.5  to  1  c.c.)  of  a  virulent  broth  culture, 
given  subcutaneously  to  a  guinea-pig,  may  produce  the  gravest  symp- 
toms and  within  six  to  eight  hours  the  animal  may  show  signs  of  great 
discomfort.  Death  occurs  usually  within  thirty-six  to  seventy-two 
hours.  Upon  autopsy  the  point  of  inoculation  is  soggy  with  serosan- 
guineous  exudate;  neighboring  lymph-nodes  are  edematous.  Lungs, 
liver,  spleen,  and  kidneys  are  congested.  There  may  be  pleuritic  and 
peritoneal  exudates.  Pathognomonic  is  a  severe  congestion  of  both 
suprarenal  bodies.  The  gastric  ulceration  recently  described  by  Rose- 
nau  and  Anderson  ^  may  occur,  but  are  by  no  means  regularly  found 
(two  out  of  fifty  in  our  series  ^). 

Determination  of  Virulence. — When  diphtheria  or  diphtheria-like 
bacilli  are  isolated  from  the  throats  of  patients  not  showing  typical 
clinical  diphtheria,  or  from  healthy  individuals  suspected  of  being 
carriers,  it  is  important  to  determine  whether  these  organisms  are  viru- 
lent. The  usual  criterion  is  their  virulence  for  guinea-pigs.  Two  c.c. 
of  a  forty-eight-hour  broth  or  ascitic  broth  culture  are  injected  sub- 
cutaneously into  a  normal  guinea-pig.  This  dose  will  kill  the  pig  in 
three  to  five  days  if  the  culture  is  virulent.  A  control  injection  should 
always  be  made  into  another  pig  of  the  same  weight,  which  has 
previously  received  an  injection  of  antitoxin  (at  least  250  units). 
Recently  Neisser  has  suggested  that  the  intracutaneous  injection  of 
the  suspected  bacilli  may  be  used  for  the  determination  of  virulence. 
This  has  the  advantage  of  economy,  as  several  tests  can  be  carried 
out  on  the  same  pig.  The  method  as  applied  by  Zingher  and  Soletsky  * 
has  been  to  use  the  following  modification  of  Neisser 's  method:  Two 
guinea-pigs  of  about  250  gr.  are  used  for  the  test.     The  abdominal 

1  Strelitz,  Arch.  f.  Kinderheilk.,  1891. 

2  Rosenau  and  Anderson,  Jour.  Inf.  Dis.,  iv,  1907. 
'  Zinsser,  Journ.  Med.  Res.,  xvii,  1907. 

*  Zingher  and  Soletsky,  Jour.  Inf.  Dis.,  1916,  xvii,  54. 


620  PATHOGENIC  MICROORGANISMS 

wall  is  prepared  by  shaving  or  plucking  out  the  hair.  A  twenty-four- 
hour  pure  culture  on  Loeffler's  medium  is  emulsified  in  20  c.c.  of 
normal  salt  solution  and  0.15  c.c.  of  this  suspension  is  injected  intra- 
cutaneously  at  a  corresponding  site  into  each  of  the  two  guinea-pigs. 
One  of  these  animals  is  given  at  the  same  time  an  intracardial  injec- 
tion of  about  250  units  of  antitoxin,  or  is  prepared  by  an  intraperi- 
toneal injection  of  antitoxin  twenty-four  hours  before  the  tests  are 
made.  Six  cultures  may  be  tested  in  this  way  on  two  animals.  Viru- 
lent strains  produce  a  definitely  circumscribed  local  infiltrated  lesion, 
which  shows  superficial  necrosis  in  two  to  three  days.  In  the  control 
pig  the  skin  remains  normal.  This  method,  in  the  hands  of  Zingher, 
gives  results  parallel  to  those  obtained  with  the  subcutaneous  tests. 

Diphtheria  Toxin.^ — Animals  and  man  infected  with  B.  diphtheriae 
show  evidences  of  severe  systemic  disturbances  and  even  organic  de- 
generations, while  the  microorganism  itself  can  be  found  in  the  local 
lesion  only.  This  fact  led  even  the  earliest  observers  to  suspect  that, 
in  part  at  least,  the  harmful  results  of  such  an  infection  were  attrib- 
utable to  a  soluble  and  diffusible  poison  elaborated  by  the  bacillus.  The 
actual  existence  of  such  a  poison  or  toxin  was  definitely  proved  by 
Roux  and  Yersin  ^  in  1889.  They  demonstrated  that  broth  cultures  in 
which  B.  diphtherias  had  been  grown  for  varying  periods  would  remain 
toxic  for  guinea-pigs  after  the  organisms  themselves  had  been  removed 
from  the  culture  fluid  by  filtration  through  a  Chamberland  filter. 

Methods  of  Production  op  Diphtheria  Toxin. — While  toxin  can 
be  produced  with  almost  all  of  the  virulent  diphtheria  bacilli,  there  is 
great  variation  in  the  speed  and  degree  of  production,  dependent  upon 
the  strain  of  organisms  employed  and  upon  the  ingredients  and  reac- 
tion of  the  medium  upon  which  they  are  grown.  Most  laboratories 
possess  one  or  several  strains  of  bacilli  which  are  empirically  known 
to  be  especially  potent  in  this  respect.  One  of  the  most  extensively 
used,  not  only  in  this  country  but  in  Europe  as  well,  is  the  strain 
known  as  ''Culture  Americana,"  or  '' Park- Williams  Bacillus  No.  8,'* 
an  organism  isolated  by  Dr.  Anna  Williams  of  the  New  York  Depart- 
ment of  Health  in  1894.  Throughout  more  than  ten  years  of  cultiva- 
tion this  bacillus  has  retained  its  great  power  of  toxin  production. 

Because  of  the  severity  of  cases  of  diphtheria  in  which  the  diph- 
theria bacilli  were  associated  with  streptococci,  many  observers  were 
led  to  believe  that  the  presence  of  streptococci  tended  to  increase  the 

1  Loeffler,  Cent,  f .  Bakt.,  1887.  2  ^ux  and  Yersin,  loc.  cit. 


BACILLUS  DIPHTHERLE  521 

toxin-producing  power  of  B.  diphtheriae.     Experiments  by  Hilbert,  ^ 
Theobald  Smith,^  and  others  seem  to  have  given  support  to  this  view. 

The  medium  most  frequently  employed  for  the  production  of  toxin 
is  a  beef-infusion  broth.  There  are  minor  differences  of  opinion  as  to 
the  most  favorable  constitution  of  this  medium  for  the  production  of 
toxin.  All  agree,  however,  in  recognizing  the  importance  of  peptone, 
without  which,  according  to  Madsen,^  no  satisfactory  toxin  has  yet  been 
produced.  This  is  added  in  proportions  of  from  one  to  two  per  cent. 
The  presence  of  sugars  in  the  medium  is  not  desirable  in  that  it  leads 
to  acid  production;  L.  Martin*  removes  the  sugars  from  the  meat  by 
fermentation  with  yeast.  Smith  ^  accomplishes  the  same  purpose  with 
B.  coli.  According  to  Park  and  Williams,^  however,  this  is  super- 
fluous, the  quantity  of  sugar  present  in  ordinary  butcher's  meat  not 
being  sufficient  to  exert  unfavorable  influence. 

Experience  has  shown  that  a  primary  alkaline  reaction  offers  the 
most  favorable  conditions  for  toxin  production.  In  all  cultures  of  B. 
diphtherise  in  non-sugar  free  broth,  there  is,  at  first,  a  production  of 
acid  and,  while  this  continues,  there  is,  as  Spronk  ^  has  shown,  little 
or  no  evidence  of  toxin  elaboration.  Park  and  Williams,^  in  an  inquiry 
into  the  question  of  reaction,  came  to  the  conclusion  that  the  best 
results  are  obtained  with  a  broth  to  which,  after  neutralization  to 
litmus,  Y  NaOH  is  added  in  an  amount  of  7  c.c.  to  the  liter.  In  such 
a  medium  the  largest  yield  of  toxin  is  obtained  after  about  five  to 
eight  days'  growth  at  a  temperature  of  37.5°  C. 

Free  access  of  oxygen  to  the  culture  medium  during  the  growth 
of  the  organisms  has  been  found  to  be  of  great  importance.  Roux  ob- 
tained this  by  passing  a  stream  of  oxygen  through  the  bouillon.  The 
supply  is  quite  sufficient  for  practical  purposes,  however,  if  the  medium 
is  distributed  in  thin  layers  in  large-necked  Erlenmeyer  flasks. 

Chemical  Nature  and  Physical  Properties  op  Diphtheria 
Toxin. — The  chemical  composition  of  diphtheria  toxin  is  not  known. 
Brieger  and  Frankel,^  by  repeated  precipitation  with  alcohol,  suc- 

1  Hilbert,  Zeit.  f.  Hyg.,  xxix,  1898.  ^  Smith,  Medical  Rec,  May,  1896. 

'  Madsen,  Kraus  und  Levaditi,  "Handbuch  d.  Technic,"  etc.,  1907. 

4  L.  Martin,  Ann.  de  Tinst.  Pasteur,  1897. 

6  Th.  Smith,  Jour.  Exp.  Med.,  iv,  1899. 

«  Park  and  Williams,  Jour.  Exp.  Med.,  1897. 

''Spronk,  Ann.  de  I'inst.  Pasteur,  1895. 

8  Park  and  Williams,  Jour.  Exp.  Med.,  1897. 

'  Brieger  und  Frankel,  Berl.  klin.  Woch.,  xi-xii,  1889. 


522  PATHOGENIC  MICROORGANISMS 

ceeded  in  extracting  from  toxic  bouillon  a  white,  water-soluble  powder 
which  possessed  most  of  the  poisonous  properties  of  the  broth  itself. 
This,  in  solution,  gave  many  of  the  useful  proteid  reactions,  but  dif- 
fered from  proteids  in  failing  to  coagulate  when  boiled  and  in  not 
giving  precipitates  when  treated  with  magnesium  sulphate,  sodium 
sulphate,  or  nitric  acid.  It  was  believed  by  them  to  be  closely  related 
to  the  albumoses,  bodies  representing  intermediate  phases  in  the  pep- 
tonization of  albumins.  Similar  results  have  been  obtained  by  Was- 
sermann  and  Proskauer,^  Brieger  and  Boer,^  and  others.  Uschinsky,^ 
on  the  other  hand,  has  disputed  the  proteid  nature  of  toxins  in  gen- 
eral, having  produced  diphtheria  toxin  by  growing  the  organism  upon 
a  medium  entirely  free  from  albuminous  bodies.  Uschinsky  believes 
that  the  protein  reactions  observed  by  others  may  be  due  to  ingredi- 
ents of  the  precipitates  other  than  the  toxin.  It  is  not  impossible, 
however,  that  the  organisms  may  have  produced  proteid  substances 
by  synthesis  from  the  simpler  substances  in  Uschinsky 's  medium.  The 
production  of  toxin  from  such  a  medium,  therefore,  is  not  a  conclusive 
argument  against  the  proteid  nature  of  toxins.  Accurate  chemical  iso- 
lation and  analysis  of  diphtheria  toxin  have  not  yet  been  accomplished. 

Diphtheria  toxin  is  destroyed,*  when  in  the  fluid  form,  by  tempera- 
tures of  58°  to  60°  C.  In  the  dry  state,  it  resists  a  temperature  of 
70°  C.  and  over,  without  change.  Light  and  free  access  of  air  produce 
rapid  deterioration.  Sealed,  protected  from  light,  and  kept  at  almost 
freezing  point,  the  toxin  remains  stable  for  long  periods.  Electrical 
currents  passed  through  toxic  broth  have  little  or  no  effect  upon  it. 

Transmission. — Diphtheria  is  transmitted  from  one  individual  to 
another  directly  or  indirectly  by  contact  or  droplet  infection — as  in 
coughing,  etc.  It  has  been  found  that  individuals  may  retain  virulent 
diphtheria  bacilli  in  nose  and  throat  for  long  periods  after  recovery 
from  the  disease.     These  are  the  so-called  "diphtheria  carriers.*' 

The  problem  of  diphtheria  carriers  has  become  one  of  considerable 
importance  and  has  been  given  special  prominence  of  recent  years  by 
the  studies  of  Von  Scholly,  Moss,  and  Nuttall  and  Graham  Smith. 
Anderson,  Goldberger  and  Hachtel  ^  studied  4,0^9  healthy  people  in 

^  Wassermann  und  Proskauer,  Deut.  med.  Woch.,  1891,  p.  585. 

2  Brieger  und  Boer,  Deut.  med.  Woch.,  1896,  p.  783. 

3  Uschinsky,  Cent.  f.  Bakt.,  xxi,  1897. 
*  Roux  et  Yersin,  loc.  cit. 

« Goldberger,  Williams  and  Hachtel,  Bull.  No.  101,  of  the  Hygienic  Laboratories, 
of  the  U.  S.  Public  Health  Service. 


BACILLUS  DIPHTHERLE  523 

the  city  of  Detroit,  and  found  that  0.928%  harhored  bacilli  identical 
morphologically  with  the  Klebs-Loeffler  bacillus.  This  figure  is  rather 
lower  than  those  of  some  other  investigators,  but  would  indicate,  as 
the  writers  stated,  that  there  were  from  5,000  to  6,000  diphtheria  car- 
riers in  the  city  of  Detroit. 

Of  19  cultures  isolated  from  19  of  the  carriers,  only  2  were  virulent, 
which  would  indicate  that  only  0.097%  of  the  people  examined  carried 
organisms  capable  of  producing  disease.  An  interesting  further  point 
is  that  the  bacillus  Hoffmanni  was  present  in  at  least  41.9%  of  over 
2,000  individuals  examined,  and  that  47  cultures,  morphologically 
identified  as  Bacillus  Hofi:manni,  were  avirulent.  This  would  confirm 
the  impression  gained,  we  believe,  by  most  experienced  laboratory 
workers  that  a  true  Hoffmanni  can  be  distinguished  with  considerable 
certainty  from  a  Klebs-Loeffler  bacillus  by  morphological  examination 
alone,  and  that  its  significance  is  probably  that  of  a  frequently  present 
saprophyte  of  the  throat  and  pharynx.  The  studies  of  Goldberger, 
Williams  and  Hachtel  also  indicate  that  in  examining  for  diphtheria 
carriers  it  is  best  not  to  confine  oneself  either  to  the  nose  or  throat,  but 
that  cultures  should  be  taken  from  both  places  in  every  ease. 

Bacteria  Similar  to  Bacillus  Diphtherise. — Bacii^lus  Hoffmanni 
{Pseudodiphtheria  hacillus). — Hoffmann- Wellenhoff,^  in  1888,  and,  at 
almost  the  same  time,  Loeffler,^  described  bacilli  which  they  had  culti- 
vated from  the  throats  of  normal  persons  and  in  several  instances  from 
those  of  diphtheritic  persons,  which  were  in  many  respects  similar  to 
true  B.  diphtheria,  but  differed  from  this  chiefly  in  being  non-patho- 
genic for  guinea-pigs.  These  organisms  were  at  first  regarded  by  some 
observers  as  merely  attenuated  diphtheria  bacilli.  More  recent  inves- 
tigations, however,  prove  them  to  be  unquestionably  a  separate  species, 
easily  differentiable  by  proper  methods.  They  differ  from  B.  diph- 
therise in  so  many  important  features,  moreover,  that  the  term  **  pseu- 
dodiphtheria bacillus*'  is  hardly  an  appropriate  one  for  them. 

Morphology. — Bacillus  Hoffmanni  is  shorter  and  thicker  than  Ba- 
cillus diphtheria.  It  is  usually  straight  and  slightly  clubbed  at  one 
end,  rarely  at  both.  Stained  with  Loeffler's  blue  it  occasionally  shows 
unstained  transverse  bands ;  unlike  B.  diphtheriae,  however,  these  bands 
hardly  ever  exceed  one  or  twofn  number  at  most.  In  many  cultures 
the  single  transverse  band  gives  the  bacillus  a  diplococcoid  appearance. 


1  Hoffmann-Wellenhoff,  Wien.  med.  Woch.,  iii,  1888. 

2  Loeffler,  Cent,  f .  Bakt.,  ii,  1887. 


624 


PATHOGENIC  MICROORGANISMS 


:?' 


S%J'-  '^ 


I 


staining. — Stained  by  Neisser's  or  Roux's  method,  no  polar  bodies 
can  be  demonstrated.  The  bacillus  forms  no  spores,  is  non-motile,  and 
possesses  no  flagella. 

Cultivation. — On  the  usual  culture  media  B.  Hoffmann!  grows  more 
luxuriantly  than  B.  diphtheriae,  developing  even  in  first  isolations  from 
the  human  body  upon  the  simple  meat-extract  media.  On  agar  plates 
its  colonies  are  larger,  less  transparent,  and  whiter  than  are  those  of 

true  diphtheria  bacilli.  In  fluid  media 
there  is  even  clouding  and  less  ten- 
dency to  the  formation  of  a  pellicle 
than  with  B.  diphtheriae.  A  positive 
means  of  distinction  between  the  two  is 
given  by  the  inability  of  B.  Hoffmanni 
to  form  acid  upon  various  sugar  media. 
The  differentiation  on  a  basis  of  acid 
formation  was  first  attempted  by  Cob- 
bett  ^  and  has  been  recently  worked  out 
systematically  by  Knapp,^  and  con- 
firmed by  various  observers.^  The  re- 
/p.    ^  «  ft.*^       /        -  r-4  ^^^^^  ^^  ^^i^  work,  carried  out  with  the 

t'     "     V^^ '    ~^^  V^  H  serum-water  media   of   Hiss,   to   which 
'■J.     ^J-.^'^      k^^      ^2  various  sugars  were  added,  show  that 
s%^  '*"■'  ,         ,,  ^jC^      1  B.  Hoffmanni  forms  acid  upon  none  of 
'  ■■'  -^>>  . ^^ — J  the   sugars   used,   while    B.    diphtheriae 

Fig.  107. — Bacillus  Hoffmanni.   acidifies  and  coagulates  media  contain- 
ing monosaccharids  and  several  of  the 
more  complex  sugars,  as  given  in  the  diagram  in  the  section  following, 
dealing  with  B.  xerosis. 

Differentiation  can  finally  be  made  on  the  basis  of  animal  patho- 
genicity, B.  Hoffmanni  being  entirely  innocuous  to  the  ordinary  labora- 
tory animals.  B.  Hoffmanni  forms  no  toxins,  and  animals  immunized 
with  it  do  not  possess  increased  resistance  to  B.  diphtherias. 

Bacillus  xerosis. — In  1884,  Kutschert  and  Neisser*  described  a 
bacillus,  isolated  from  the  eyes  of  patients  suffering  from  a  form  of 
chronic  conjunctivitis  known  as  xerosis.  This  bacillus,  which,  morpho- 
logically, is  almost  identical  with  B.  dif)htheriae,  they  believed  to  be  the 
etiological  factor  of  the  disease.    The  frequency  with  which  it  has  been 

1  Cohbett,  Cent.  f.  Bakt.,  1898.        ^  Knapp,  Jour.  Med.  Res.,  vii,  1904. 

'  Graham  Smith,  Jour,  of  Hyg.,  vi,  1906;  Zinsser,  Jour.  Med.  Res.,  xvii,  1907. 

*  Kutschert  und  Neisser,  Deut.  med.  Woch.,  xxiv,  1884. 


BACILLUS  DIPHTHERLE 


525 


isolated  from  normal  eyes,  precludes  this  etiological  relationship,  and 
it  may  safely  be  regarded  as  a  harmless  parasite  which  may  indeed  be 
more  abundant  in  the  slightly  inflamed  than  in  the  normal  conjunctiva. 

Morphology. — B*  xerosis  resembles  B.  diphtherias  closely.  It  is 
occasionally  shorter  than  this,  but  on  the  whole  no  absolute  morpho- 
logical differentiation  between  the 
two  is  possible.  It  forms  no  spores 
and  is  non-motile.  Polar  bodies 
may  occasionally  be  seen. 

Cultivation.  —  On  Loeffler's 
Mood  serum,  on  agar,  glycerin 
agar,  and  in  hroth,  its  growth  is 
very  similar  to  that  of  B.  diph- 
therias, but  more  delicate  through- 
out. It  cannot  easily  be  cultivated 
upon  the  simple  meat-extract 
media,  nor  will  it  grow  on  gelatin 
at  room  temperature.  Its  colonies 
on  glycerin  or  glucose  agar  are 
microscopically  identical  with 
those  of  B.  diphtherise. 

Differentiation. — It  differs 
from  B.  diphtherias  distinctly  in 
its    acidifying    action    on    sugar 

media.  These  relations  were  first  worked  out  by  Knapp  for  various 
sugars  and  the  alcohol  mannit,  and  have  been  extensively  confirmed 
by  others.    See  table  on  page  526. 

A  reference  to  the  table  shows  that  differentiation  may  be  made 
by  the  use  of  two  sugars — saccharose  and  dextrin.  B.  diphtherias 
forms  acid  from  dextrin,  not  from  saccharose ;  B.  xerosis  from  saccha- 
rose, not  from  dextrin ;  B.  Hoffmanni  does  not  form  acid  from  either. 

B.  xerosis  is  non-pathogenic  to  animals  and  forms  no  toxin. 

The  Diphtheroid  Bacilli. — In  addition  to  the  bacteria  mentioned 
above,  there  is  a  large  group  of  microorganisms  spoken  of  as  the  diph- 
theroid bacilli,  largely  because  of  their  morphological  resemblance  to 
the  diphtheria  bacillus.  For  this  group,  Lehman  and  Neumann  have 
suggested  the  term  corynehacterium.  The  characteristics  of  this  group 
are  a  morphological  similarity  to  the  diphtheria  bacillus,  that  they  are 
Gram-positive,  non-motile,  often  show  metachromatic  granules  and 
have  no  spores.    It  is  not,  at  the  present  writing,  possible  to  formulate 


Fig.   108. — Colonies  op  Bacillus 
Hoffmanni  on  Agar. 


526 


PATHOGENIC  MICROORGANISMS 


a  classification  of  these  organisms.    They  are  apparently  very  numer- 
ous and  have  been  isolated  from  a  great  many  different  sources,  both 


Hiss  serum-water  media  plus  1% 

B.  Diphtheriae 

B.  Xerosis 

B.  Hoffmanni 

Dextrose 

+ 
+ 

+ 
+ 

Saccharose 

_ 

Dextrin 

_ 

^ 


« 


gH 


'-^  ^<S^\ 


\^\, 


Fig.  109. — ^Bacillus  xerosis. 


in  connection  with  the  human  body  and  in  nature.  Recently  Bunting 
and  Yates  have  claimed  that  an  organism  of  this  group  has  etiological 
connection  with  Hodgkin^s  disease.  Studies  by  many  other  workers, 
notably  by  Bloomfield  and  Fox,  and  studies  going  on  in  our  own 

laboratory  show  that  organisms  very 
similar  to  these  strains  can  be  isola- 
ted from  the  skin,  from  the  lymph 
nodes  of  healthy  and  diseased  people, 
from  ascitic  fluid  in  varying  condi- 
tions, and  from  supposedly  sterile 
tissues.  They  are  frequently  present 
in  the  nasal  mucus  and  in  the  throat, 
and  are  so  ubiquitous  that  any  asso- 
ciation of  them  with  specific  disease 
must  be  very  conservatively  ap- 
proached. 
Very  similar  to  this  group  are  the  bacilli  of  pseudo-tuberculosis 
ovis,  isolated  from  necrotic  lesions  in  the  kidneys  of  sheep  by  Preisz 
and  Nocard.  It  is  impossible  at  present  to  do  more  than  indicate  that 
the  '' diphtheroid  bacilli*'  are  a  large  heterogeneous  group,  held  to- 
gether by  morphological  and  superficial  cultural  similarity  and  largely 
consisting  of  saprophytes  and  probably  harmless  parasites  on  the  hu- 
man and  animal  body. 

Shick  Reaction. — The  studies  of  Roemer  and  others  have  shown 
that  the  blood  of  the  majority  of  normal  adults  contains  a  small 
amount  of  diphtheria  antitoxin.  This  normal  antitoxin  probably  ac- 
counts for  resistance  of  many  individuals  to  diphtheria.  Its  presence 
may  be  very  easily  detected  by  means  of  the  Shick  reaction.  A  stand- 
ardized diphtheria  toxin  is  diluted  in  normal  saline  so  that  0.1  c.c.  of 
the  solution  contains  1/50  M.L.D.  for  a  guinea-pig.  This  amount  is 
injected  intracutaneously.  If  the  blood  of  the  subject  has  less  than 
1/30  unit  of  antitoxin  per  c.c,  a  positive  reaction  appears  in  twenty- 
four  to  thirty -six  hours,  which  consists  in  a  slight  infiltration  of  skin 


BACILLUS  DIPHTHERIA  527 

surrounded  by  a  red  areola,  one  to  two  cm.  in  diameter.  A  negative 
reaction  indicates  that  sufficient  natural  antitoxin  is  present  to  pro- 
tect the  individual  against  diphtheria,  although  he  may  nevertheless 
harbor  the  bacilli  as  a  carrier.  It  has  been  found  unnecessary  to  give 
prophylactic  injections  of  antitoxin  to  individuals  with  a  negative 
Shick  reaction.  Park  and  Zingher  ^  have  found  that  negative  reactions 
were  obtained  in  93  per  cent  of  the  new-born,  and  became  less  fre- 
quent up  to  the  second  or  fifth  year,  when  37  per  cent  were  negative. 
In  older  children  negative  reactions  were  more  frequently  met  with, 
and  about  90  per  cent  of  adults  were  negative.  A  pseudoreaction 
which  appears  earlier  than  the  true  reaction  and  which  disappears  in 
twenty-four  to  forty-eight  hours  is  occasionally  seen  in  individuals 
who  have  natural  antitoxin.  This  is  due  to  sensitiveness  to  some  of 
the  proteins  used  in  the  injection,  and  may  be  reproduced  in  the  same 
individual  by  the  injection  of  autolysate  of  diphtheria  bacilli  or  some- 
times by  the  injection  of  broth  media.  The  most  satisfactory  method 
for  detection  of  the  pseudoreaction  is  to  make  a  control  injection  of 
1/50  M.L.D.  of  a  toxin  which  has  been  heated  at  80°  C.  for  five 
minutes.  This  heating  destroys  the  toxin,  but  leaves  uninjured  the 
substances  which  produce  the  pseudoreaction. 

Active  Immunization  with  Neutralized  Diphtheria  Toxin. — It  was 
suggested  some  years  ago  by  Theobald  Smith  that  more  lasting  prophy- 
lactic immunity  in  diphtheria  might  be  produced  if  individuals  were 
stimulated  to  produce  their  own  antitoxin  by  injection  of  toxin-anti- 
toxin mixture.  This  method  has  recently  been  applied  by  Behring  and 
by  Park  and  Zingher. ^  It  has  been  found  that  individuals  who  have 
no  natural  antitoxin,  as  shown  by  a  positive  Shick  reaction,  may  be 
rendered  Shick-negative  by  injection  of  diphtheria  toxin  partially  neu- 
tralized by  antitoxin.  For  this  purpose  Park  and  Zingher  recommend 
three  injections  at  intervals  of  six  or  seven  days,  consisting  of  1  c.c. 
of  toxin-antitoxin  mixture  plus  1,000,000,000  killed  diphtheria  bacilli. 
The  mixture  is  made  up  to  contain  85  to  90  per  cent  of  an  L.  -j-  dose 
of  toxin,  per  unit  of  antitoxin.  The  toxin  used  contains  about  four 
L.  +  doses  per  c.c.  Individuals  treated  in  this  way  develop  antitoxin 
slowly,  and  in  many  instances  their  Shick  reaction  does  not  become 
negative  for  from  six  to  eight  months  after  immunization.  A  few  indi- 
viduals apparently  do  not  respond  to  such  immunization  by  the  devel- 
opment of  antitoxin  and  remain  Shick-positive. 

1  Park  and  Zingher,  Am.  Jour.  Pub.  Health,  1916,  vi,  p.  431. 

2  Park  and  Zingher j  Jour.  Am.  Med.  Ass.,  1915,  Ixv,  p.  2216. 


CHAPTER  XXXVI 
BACILLUS    MALLEI 
{Glanders  Bacillus) 

Glanders  is  an  infectious  disease  prevalent  chiefly  among  horses, 
but  transmitted  occasionally  to  other  domestic  animals  and  to  man. 
The  microorganism  causing  the  disease,  though  seen  and  described  by 
several  earlier  authors,  was  first  obtained  in  pure  culture  and  accurately 
studied  by  LoeflEler  and  Schiitz  ^  in  1882. 

Morphology  and  Staining. — The  glanders  bacillus  or  B.  mallei  is  a 
rather  smaU  rod  with  rounded  ends.^  Its  length  varies  from  3  to  4 
micra,  its  breadth  from  0.5  to  0.75  micron.  Variation  in  size  be- 
tween separate  individuals  in  the  same  culture  is  characteristic.  The 
rods  are  usually  straight,  but  may  show  a  slight  curvature.  The  bacillus 
is  non-motile.  There  are  no  flagella  and  no  spores  are  formed.  The 
grouping  of  the  bacilli  in  smears  shows  nothing  very  characteristic. 
Usually  they  appear  as  single  bacilli  lying  irregularly  parallel,  often  in 
chains  of  two  or  more.  In  old  cultures,  involution  forms  appear  which 
are  short,  vacuolated,  and  almost  coccoid. 

While  the  glanders  bacillus  stains  rather  easily  with  the  usual  anilin 
dyes,  it  is  so  easily  decolorized  that  especial  care  in  preparing  specimens 
must  be  observed.  Stained  in  the  usual  manner  with  methylene-blue, 
it  shows  marked  irregularity  in  its  staining  qualities;  granular,  deeply 
staining  areas  alternating  with  very  faintly  stained  or  entirely  unstained 
portions.  This  diagnostic  ally  helpful  characteristic  has  been  variously 
interpreted  as  a  mark  of  degeneration  or  a  preparatory  stage  for  sporula- 
tion.  It  is  probably  neither  of  the  two,  but  an  inherent  irregularity  in 
the  normal  protoplasmic  composition  of  the  bacillus,  not  unlike  that 
cf  B.  diphtherise.  The  bacillus  is  decolorized  by  Gram's  method  of 
staining. 

Cultivation. — The  glanders  bacillus  is  easily  grown  on  all  of  the 

1  Loeffler  und  Schiitz,  Deut.  med.  Woch.,  1882. 

2  Loeffler,  Arb.  a.  d.  kais.  Gesundheitsamt,  1886. 

528 


BACILLUS  MALLEI  529 

usual  meat-infusion  media.  It  is  practically  indifferent  to  moderate 
variations  in  reaction,  growing  equally  well  upon  neutral,  slightly  acid, 
or  slightly  alkahne  culture  media.  Glycerin  or  small  quantities  of 
glucose  added  to  media  seem  to  render  them  more  favorable  for  the 
cultivation  of  this  bacillus. 

Upon  agar  the  colonies  show  little  that  is  characteristic.  They 
appear  after  twenty-four  hours  at  37.5°  C.  as  yellowish-white  spots, 
at  first  transparent,  later  more  opaque.     They  are  round,  with  an  even 


^' 


Fig,  110. — Glanders  Bacillus.    From  potato  culture.    (After  Zettnow.) 

border,  and  microscopically  appear  finely  granular.  The  older  the  cul- 
tures are,  the  more  yellow  do  they  appear. 

On  gelatin  at  room  temperature,  growth  is  slow,  grayish- white,  and 
no  liquefaction  of  the  gelatin  occurs.  Growth  upon  this  medium,  is 
never  abundant. 

In  broth,  there  is,  at  first,  diffuse  clouding,  later  a  heavy,  tough, 
slimy  sediment  is  formed.  At  the  same  time  the  surface  is  covered  with 
a  similarly  slimy  pelHcle.  The  broth  gradually  assumes  a  dark  brown 
color. 

In  milk,  coagulation  takes  place  slowly.  In  litmus  milk,  acidifica- 
tion is  indicated. 

The  growth  upon  potato  presents  certain  features  which  are  diagnos- 
tically  valuable.  On  potatoes  which  are  not  too  acid  growth  is  abundant 
and  within  forty-eight  hours  covfers  the  surface  as  a  yellowish,  trans- 


530  PATHOGENIC  MICROORGANISMS 

parent,  slimy  layer.  This  gradually  grows  darker  until  it  has  assumed 
a  deep  reddish-brown  hue.  In  using  this  feature  of  the  growth  diagnos- 
tically,  it  must  not  be  forgotten  that  a  very  similar  appearance  upon 
potato  occurs  in  the  case  of  B.  pyocyaneus. 

Biological  Considerations. — Bacillus  mallei  is  aerobic.^  Growth  under 
anaerobic  conditions  may  take  place,  but  it  is  slow  and  impoverished. 
The  most  favorable  temperature  for  its  cultivation  is  37.5°  C.  It 
fails  to  develop  at  temperatures  below  22°  C.  or  above  43°  C.  On 
artificial  media,  if  kept  cool  and  in  the  dark,  and  in  sealed  tubes,  the 
glanders  bacillus  will  retain  its  viability  for  months  and  years.  On 
gelatin  and  in  bouillon,  it  lives  for  a  longer  time  than  on  the  other  media. 
Exposed  to  strong  sunlight  it  is  killed  within  twenty-four  hours.  Heat- 
ing to  60°  C.  kills  it  in  two  hours,  to  75°  C.  within  one  hour.  Thorough 
drying  kills  the  glanders  bacillus  in  a  short  time.  In  water,  under  the 
protected  conditions  that  are  apt  to  prevail  in  watering-troughs,  the 
bacillus  may  remain  alive  for  over  seventy  days.  The  resistance  to 
chemical  disinfectants  is  not  very  high.^  Carbolic  acid,  one  per  cent, 
kills  it  in  thirty  minutes,  bichlorid  of  mercury,  0.1  per  cent,  in  fifteen 
minutes. 

Pathogenicity. — Spontaneous  infection  with  the  glanders  bacillus 
occurs  most  frequently  in  horses.  It  occurs  also  in  asses,  in  cats,  and, 
more  rarely,  in  dogs.  In  man  the  disease  is  not  infrequent  and  is 
usually  contracted  by  those  in  habitual  contact  with  horses.  Experi- 
mental inoculation  is  successful  in  guinea-pigs  and  rabbits.  Cattle, 
hogs,  rats,  and  birds  are  immune  to  experimental  and  spontaneous 
infections  alike. 

Spontaneous  infection  takes  place  by  entrance  through  the  broken 
skin,  through  the  mucosa  of  the  mouth  or  nasal  passages.  Infection  in 
horses  not  infrequently  takes  place  through  the  digestive  tract.^  In  all 
cases,  so  far  as  we  know,  previous  injury  to  either  the  skin  or  to  the 
mucosa  is  necessary  for  penetration  of  the  bacilli  and  the  development 
of  the  disease. 

Glanders  in  horses  may  occur  in  an  acute  or  chronic  form,  depending 
upon  the  relative  virulence  of  the  infecting  culture  and  the  susceptibility 
of  the  subject.  The  more  acute  form  of  the  disease  is  usually  limited 
to  the  nasal  mucosa  and  upper  respiratory  tract.  The  more  chronic 
type  of  the  disease  is  often  accompanied  by  multiple  swellings  of  the 

^  Loeffler,  loc.  cit.  ^  i^rn^er,  Ziegler's  Beitr.,vi,  1889. 

3  Nocard,  Bull,  de  la  soc.  centr.  de  m6d.  v6t.,  1894. 


BACILLUS  MALLEI 


531 


skin  and  general  lymphatic  enlargement.     This  form  is  often  spoken  of 
as  "farcy." 

Acute  glanders  in  the  horse  begins  violently  with  fever  and  prostra- 
tion. After  two  or  three  days  there  is  a  nasal  discharge,  at  first  serous, 
later  seropurulent.  At  the  same  time  there  is  ulceration  of  the  nasal 
mucosa  and  acute  swelling  of  the  neighboring  lymph  nodes.  These 
may  break  down  and  form  deep  pus-discharging  sinuses  and  ulcers. 


(  •     3 


.'^■* 


;^ 


Fig.  ill. — Glanders  Bacilli  in  Tissue.      (From  a  drawing  furnished  by 

Dr.  James  Ewing.) 


Finally,  there  is  involvement  of  the  lungs  and  death  within  four  to  six 
weeks. 

When  the  disease  takes  the  chronic  form  the  onset  is  more  gradual. 
Concomitant  with  the  nasal  inflammation  there  is  a  formation  of  subcu- 
taneous swellings  all  over  the  body,  some  of  which  show  a  tendency  to 
break  down  and  ulcerate.  Together  with  this  the  lymphatics  all  over 
the  body  become  enlarged.  The  disease  may  last  for  several  years,  and 
occasionally  may  end  in  complete  cure.     In  horses  the  chronic  form  of 


532  PATHOGENIC   MICROORGANISMS 

the  disease  is  by  far  the  more  frequent.  In  man  the  disease  is  similar 
to  that  of  the  horse  except  that  the  point  of  origin  is  more  frequently 
in  some  part  of  the  skin  rather  than  in  the  nasal  mucosa,  and  the 
clinical  symptoms  differ  accordingly.  The  onset  is  usually  violent, 
with  fever  and  systemic  symptoms.  At  the  point  of  infection  a  nodule 
appears,  surrounded  by  lymphangitis  and  swelling.  A  general  papular 
eruption  may  occur.  The  papules  may  become  pustular,  and  the 
clinical  features  may  thus  simulate  variola.  This  type  of  the  disease 
usually  ends  fatally  in  eight  to  ten  days.  The  chronic  form  of  the 
disease  in  man  is  much  like  that  in  the  horse,  but  is  more  frequently 
fatal. 

The  histological  appearance  of  the  glanders  nodules  is  usually  one  of 
diffuse  leucocytic  infiltration  and  the  formation  of  young  connective 
tissue  which  preponderates  more  and  more  as  the  disease  becomes 
chronic.  Virchow  has  classed  these  lesions  with  the  granulomata. 
From  the  center  of  such  nodules  B.  mallei  may  often  be  obtained  in  pure 
culture.  The  nodules  may  be  generally  distributed  throughout  the 
internal  organs.  The  bacilli  themselves  are  found,  apart  from  the 
nodules,  in  the  nasal  secretions,  and  occasionally  in  the  circulating 
blood.^ 

The  bacteriological  diagnosis  of  glanders  may  be  made  by  isolating 
and  identifying  the  bacilli  from  any  of  the  above-mentioned  sources. 
When  superficial  nodules  can  be  opened  for  the  purpose  of  diagnosis  this 
may  prove  an  easy  task.  The  most  diagnostic  ally  helpful  medium  in 
such  cases  is  potato.  In  a  majority  of  cases,  however,  isolation  is  ex- 
tremely difficult  and  resort  must  be  had  to  animal  inoculation.  The 
most  suitable  animal  for  this  purpose  is  the  male  guinea-pig.  Intra- 
peritoneal inoculation  of  such  animals  with  material  containing  glanders 
bacilli  leads  within  two  or  three  days  to  tumefaction  and  purulent 
inflammation  of  the  testicles.  Such  an  experiment,  spoken  of  as  the 
"Strauss  test,"  ^  should  always  be  reinforced  by  cultural  examination 
of  the  testicular  pus,  the  spleen,  and  the  peritoneal  exudate  of  the 
animals  employed. 

Toxin  of  Bacillus  mallei. — The  toxin  of  B.  mallei,  or  mallein,  belongs 
to  the  class  of  endotoxins.  The  toxic  products  have  been  invariably 
obtained  by  extraction  of  dead  bacilli.^  Mallein  differs  from  many 
other  bacterial  poisons  in  being  extremely  resistant.     It  withstands 

1  Wassilieff,  Deut.  med.  Woch.,  1883. 

^  StraiLSS,  Arch,  de  m^d.  exp.,  1889. 

» Kresling,  Arch.  d.  sci.  bioL,  1892;  Preuser,  Berl.  thierarzt.  Woch.,  1894. 


BACILLUS  MALLEI  533 

temperatures  of  120°  C.  and  prolonged  storage  without  noticeable  loss  of 
strength.* 

In  its  physiological  action  upon  healthy  animals,  mallein  is  not  a 
powerful  poison.  It  can  be  given  in  considerable  doses  without  causing 
death.  Mallein  may  be  obtained  by  a  variety  of  methods.  Helman 
and  Kalning,  the  discoverers  of  this  toxin,  used  filtered  aqueous  and 
glycerin  extracts  of  potato  cultures.  Roux  ^  cultivates  virulent  gland- 
ers bacilli  in  flasks  containing  250  c.c.  each  of  5  per  cent  glycerin 
bouillon.  Growth  is  allowed  to  continue  at  35°  C.  for  one  month.  At 
the  end  of  this  time,  the  cultures  are  sterilized  at  100°  for  thirty  min- 
utes, and  evaporated  on  a  water  bath  to  one-tenth  their  original  volume. 
They  are  then  filtered  through  paper.  This  concentrated  poison  is 
diluted  ten  times  with  0.5  per  cent  carbolic  acid  before  use.  Concen- 
tration is  done  merely  for  purposes  of  conservation.  The  diagnostic 
dose  of  such  mallein  for  a  horse  is  0.25  c.c.  of  the  undiluted  fluid. 

At  the  Washington  Bureau  of  Animal  Industry,  mallein  is  prepared 
by  growing  the  bacilli  for  five  months  at  37.5°  C.  in  glycerin-bouillon. 
This  is  then  boiled  for  one  hour  and  allowed  to  stand  in  a  cool  place 
for  one  week.  The  supernatant  fluid  is  then  decanted  and  filtered 
through  clay  filters  by  means  of  a  vacuum  pump.  The  filtrate  is 
evaporated  to  one-third  its  original  volume  on  a  water  bath,  and  the 
evaporated  volume  resupplied  by  a  1  per  cent  carboHc  acid  solution 
containing  about  10  per  cent  of  glycerin. 

Diagnostic  Use  of  Mallein. — The  injection  of  a  proper  dose  of 
mallein  into  a  horse  suffering  from  glanders  is  followed  within  six  to 
eight  hours  by  a  sharp  rise  of  temperature,  often  reaching  104°  to  106°  F. 
(40°  C.  +).  The  high  temperature  continues  for  several  hours  and  then 
begins  gradually  to  fall.  The  normal  is  not  usually  regained  for  several 
days.  Locally,  at  the  point  of  injection,  there  appears  within  a  few 
hours  a  firm,  hot,  diffuse  swelling,  which  gradually  extends  until  it  may 
cover  areas  of  20  to  30  centimeters  in  diameter.  The  swelhng  is  in- 
tensely tender  during  the  first  twenty-four  hours,  and  lasts  for  three  to 
nine  days.  Together  with  this  there  are  marked  symptoms  of  general 
intoxication.  In  normal  animals  the  rise  of  temperature  following  an 
injection  is  trifling,  and  the  local  reaction  is  much  smaller  and  more 
transient.  Injections  are  best  made  into  the  breast  or  the  side  of  the 
neck. 

1  Wladimiroff,  in  Kraus  und  Levaditi,  "  Handbuch,"  etc.,  1908- 

2  Roux  et  Nocard.  Bull.  d.  1.  soc.  centr.  vet.,  1892- 


534  PATHOGENIC  MICROORGANISMS 

The  directions  given  by  the  United  States  Government  for  using 
mallein  for  the  diagnosis  of  glanders  in  horses  are  as  follows : 

"Make  the  test,  if  possible,  with  a  healthy  horse,  as  well  as  with 
one  or  more  affected  or  supposed  to  be  affected  with  glanders.  Take 
the  temperature  of  all  these  animals  at  least  three  times  a  day  for  one 
or  more  days  before  making  the  injections. 

"The  injection  is  most  conveniently  made  at  6  or  7  o^clock  in  the 
morning,  and  the  maximum  temperature  will  then  usually  be  reached 
by  or  before  10  p.m.  of  the  same  day. 

"Use  for  each  horse  one  cubic  centimeter  of  the  mallein  solution  as 
sent  out,  and  make  the  injection  beneath  the  skin  of  the  middle  of  one 
side  of  the  neck,  where  the  local  swelling  can  be  readily  detected. 

"Carefully  sterilize  the  syringe  after  injecting  each  horse  by  flaming 
the  needle  over  an  alcohol  lamp  or,  better,  use  separate  syringes  for 
healthy  and  suspected  animals.  If  the  same  syringe  is  used,  inject  the 
healthy  animals  first,  and  flame  the  needle  of  the  syringe  after  each 
injection. 

"Take  the  temperature  every  two  hours  for  at  least  eighteen  hours 
after  the  injection.  Sterilize  the  thermometer  in  a  5  per  cent  solu- 
tion of  carbolic  acid,  or  a  0.2  per  cent  solution  of  corrosive  sublimate, 
after  taking  the  temperature  of  each  animal. 

"  The  temperature,  as  a  rule,  will  begin  to  rise  from  four  to  eight  hours 
after  the  injection,  and  reach  its  maximum  from  ten  to  sixteen  hours 
after  injection.  On  the  day  succeeding  the  injection  take  the  tempera- 
ture at  least  three  times. 

"  In  addition  to  the  febrile  reaction,  note  the  size,  appearance,  and 
duration  of  any  local  swelling  at  the  point  of  injection.  Note  the  general 
condition  and  symptoms  of  the  animal,  both  before,  during,  and  after 
the  test. 

"  Keep  the  solution  in  the  sealed  bottle  and  in  a  cool  place,  and  do 
not  use  it  when  it  is  clouded  or  if  it  is  more  than  six  weeks  old  *  when  it 
leaves  the  laboratory  of  the  Bureau  it  is  sterile." 

If  the  result  of  first  injection  is  doubtful,  the  horse  should  be 
isolated  and  retested  in  from  one  to  three  months,  when  the  slight 
immunity  conferred  by  the  first  injection  will  have  disappeared. 
The  second  injection  into  healthy  horses  usually  shows  no  reaction 
whatever. 

Mallein  may  cause  reactions  in  the  presence  of  other  diseases  than 
glanders,  such  as  bronchitis,  periostitis,  and  other  inflammatory  lesions 
and  is  not  so  specifically  valuable  as  tuberculin  for  diagnosis. 


BACILLUS  MALLEI  635 

Complement  Fixation  in  Glanders. — Diagnostic  complement  fixation 
for  the  diagnosis  of  glanders  has  been  developed  by  McNeil  and  01m- 
stead  at  the  New  York  Department  of  Health.  The  antigen  is  made 
by  growing  the  glanders  bacilli  on  a  1,6  per  cent  glycerin  potato  agar. 
From  this  stock  cultures  transplanted  are  made  upon  a  neutral  meat- 
free-veal  peptone  agar.  Twenty-four-hour  growths  are  washed  off  with 
distilled  water  sterilized  at  80°  C.  for  four  hours  and  filtered  through 
a  Berkefeld.  After  filtration  the  antigen  must  again  be  sterilized  at 
80°  for  one  hour. 

Immunity. — Recovery  from  a  glanders  infection  does  not  confer 
immunity  against  a  second  inoculation.^  Artificial  active  immuniza- 
tion has  been  variously  attempted  by  treatment  with  attenuated  cul- 
tures, with  dead  bacilli,  and  with  mallein,  but  without  convincing 
results. 

The  serum  of  subjects  suffering  from  glanders  contains  specific 
agglutinins.^  These  are  of  great  importance  diagnostically  if  the  tests 
are  made  with  dilutions  of,  at  least,  1  in  500,  since  normal  horse  serum 
may  agglutinate  B.  mallei  in  dilutions  lower  than  this. 

*  Finger,  Ziegler's  Beitrage,  vi,  1899.        ^  Galtier,  Jour,  de  med.  vet.,  1901. 


CHAPTER  XXXVII 
BACILLUS   INFLUENZA   AND   CLOSELY   RELATED   BACTERIA 

There  is  no  other  epidemic  disease  which  spreads  over  such  enormous 
territories,  and  with  such  speed,  as  influenza.  Epidemics  have  been 
numerous  and  reports  of  the  disease,  unquestionably  recognizable,  are 
extant  even  from  the  most  remote  times.  The  last  serious  epidemic 
occurred  in  the  years  1889  to  1890,  when  the  disease,  spreading  from 
the  East,  traveled  through  Russia  and,  pandemically,  attacked  all  of 
Europe,  then  reached  America,  and  eventually,  having  traveled  east- 
ward as  well  as  westward  from  its  point  of  origin,  became  prevalent 
in  China,  Japan,  Australia,  and  Africa.  Hundreds  of  thousands  were 
attacked  and  the  mortality  of  this  epidemic  was  high.  Its  enormous 
scope  and  the  rapidity  of  its  spread  were  facilitated  probably  by  the 
activity  of  modern  international  commerce. 

The  character  of  the  disease  pointed  so  definitely  to  a  bacterial 
etiology  that  numerous  attempts  to  isolate  a  specific  microorganism 
were,  of  course,  made.  Pfeiffer  ^  finally,  in  1892,  described  the  bacillus 
which  is  at  present  definitely  recognized  as  the  etiological  factor  of 
influenza. 

Morphology  and  Staining. — The  bacillus  of  influenza"  (Pfeiffer  bacillus) 
is  an  extremely  small  organism,  about  0.5  micron  long  by  0.2  to  0.3 
micron  in  width.  They  are  somewhat  irregular  in  length,  but  show 
rounded  ends.  They  rarely  form  chains.  They  are  non-motile,  and 
do  not  form  spores. 

Influenza  bacilli  stain  less  easily  than  do  most  other  bacteria  with 
the  usual  anilin  dyes,  and  are  best  demonstrated  with  10  per  cent 
aqueous  fuchsin  (5  to  10  minutes),  or  with  Loeffler's  methylene-blue 
(5  minutes).  They  are  Gram-negative,  giving  up  the  anilin-gentian- 
violet  stain  upon  decolorization.  Occasionally  slight  polar  staining 
may  be  noticed.  Grouping,  especially  in  thin  smears  of  bronchial 
secretion,  is  characteristic,  in  that  the  bacilli  very  rarely  form  threads 

1  Pfeiffer,  Deut.  med.  Woch.,  ii,  1892;  Zeit.  f.  Hyg.,  xiii,  1892;  Pfeiffer  und  Beok, 
Deut.  med.  Woch.,  xxi,  1893. 

536 


BACILLUS  INFLUENZA 


537 


or  chains,  usually  lying  together  in  thick,  irregular  clusters  without 
definite  parallelism. 

Isolation  and  Cultivation. — Isolation  of  the  influenza  bacillus  is 
not  easy.  Pfeiffer  ^  succeeded  in  growing  the  bacillus  upon  serum-agar 
plates  upon  which  he  had  smeared  pus  from  the  bronchial  secretions  of 
patients.     Failure  of  growth  in  attempted  subcultures  made  upon  agar 


Fig.  112. — Bacillus  influenza.    Smear  from  pure  culture  on  blood  agar. 


and  gelatin,  however,  soon  taught  him  that  the  success  of  his  first  culti- 
vations depended  upon  the  ingredients  of  the  pus  carried  over  from  the 
sputum.  Further  experimentation  then  showed  that  it  was  the  blood, 
and  more  particularly  the  hemoglobin,  in  the  pus  which  had  made  growth 
possible  in  the  first  cultures.     Pfeiffer  made  his  further  cultivations 

1  Pfeiffer,  loc.  cic. 


538 


PATHOGENIC   MICROORGANISMS 


upon  agar,  the  surface  of  which  had  been  smeared  with  a  few  drops  of 
blood  taken  sterile  from  the  finger.  Hemoglobin  separated  from  the 
red  blood  cells  was  found  to  be  quite  as  efficient  as  whole  blood.  This 
method  of  Pfeiffer  is  still  the  one  most  frequently  employed  for  isolation 
and  cultivation.  Whole  blood  taken  from  the  finger  may  be  either 
smeared  over  the  surface  of  slants  or  plates,  or  mixed  with  the  melted 
meat-infusion  agar.  In  isolating  from  sputum,  only  that  secretion  should' 
be  used  which  is  coughed  up  from  the  bronchi  and  is  uncontaminated 
by  microorganisms  from  the  mouth.  It  may  be  washed  in  sterile  water 
or  bouillon  before  transplantation,  to  remove  the  mouth  flora  adhe- 


:\^r 


H,  s\*  K, 


^V^l^ 


Fig.  113. — Bacillus  influenza.      Smear  from  sputum.     (After  Heim.) 


rent  to  the  outer  surface  of  the  little  clumps  of  pus.  The  blood  of 
pigeons  or  that  of  rabbits  may  be  substituted  for  human  blood.  The 
former  seems  to  be  the  more  favorable  of  the  two  and  even  more  so  than 
human  blood.  Pigeons  may  be  easily  bled  for  this  purpose  from  the 
large  veins  under  the  wing.  Huber*  has  succeeded  in  cultivating  in- 
fluenza bacilli  upon  media  containing  a  soluble  hemoglobin  derivative 
known  as  hematogen.  This  substance,-  however,  offers  some  difficulties 
to  sterilization  and  is  not  so  favorable  as  whole  blood.  The  absence 
of  oxyhemoglobin  from  the  hematogen,  however,  is  theoretically  im- 
portant in  that  it  shows  that  hemoglobin  is  suitable  for  the  growth 

1  Huber,  Zeit.  f.  Hyg.,  xv,  1893. 


BACILLUS  INFLUENZA 


539 


of  this  bacillus  because  of  its  nutrient  qualities  and  not  by  virtue 
of  its  oxygen-carrying  properties.  Although  the  presence  of  hemo- 
globin seems  to  be  a  necessity  for  the  successful  cultivation  of  the 
bacillus,  the  quantity  present  need  not  be  very  large.  Ghon  and  Preyss  ^ 
showed  that  an  amount  too  small  to  be  demonstrated  spectroscopically 
sufficed  for  its  growth. 

Other  substances  which,  added  to  neutral  or  slightly  alkaline  agar,t. 
have  been  used  for  the  cultivation  of  influenza  bacilli  are  the  yolk  of 
eggs  2  (not  confirmed)  and  spermatic  fluid .^     None  of  these,  however, 
is  as  useful  as  the  blood  media.     Symbiosis  with  staphylococci,'*  too, 


Fig.  114. — Colonies  of  Influenza  Bacillus  on  Blood  Agae.    (After  Heim.) 

has  been  found  to  create  an  environment  favorable  for  their  develop- 
ment. 

Influenza  bacilli  do  not  grow  at  room  temperature.  Upon  suitable 
media  at  37.5°  C.  colonies  appear  at  the  end  of  eighteen  to  twenty-four 
hours,  as  minute,  colorless,  transparent  droplets,  not  unlike  spots  of 
moisture.  These  never  become  confluent.  The  limits  of  growth  are 
reached  in  two  or  three  days.     To  keep  the  cultures  alive,  tubes  should 


1  Ghon  und  Preyss,  Cent.  f.  Bakt.,  xxxv,  1904. 
8  Nastjukoff,  Cent.  f.  Bakt.,  Ref.,  xix,  1896. 
*Cantani,  Cent.  f.  Bakt.,  xxii,  1897. 
*  Grassberger,  Zeit.  f.  Hyg.,  xxv,  1897. 


540  .  PATHOGENIC  MICROORGANISMS 

be  stored  at  room  temperature  and  transplantations  done  at  intervals 
not  longer  than  four  or  five  days. 

Biology. — ^The  bacillus  is  aerobic,  growing  in  broth-blood  mixtures 
only  upon  the  surface,  hardly  at  all  in  agar  stab  cultures,  and  not  at  all 
under  completely  anaerobic  conditions. 

As  it  does  not  form  spores,  it  is  exceedingly  sensitive  to  heat,  desicca- 
tion, and  disinfectants.^  Heating  to  60°  C.  kills  the  bacilli  in  a  few  min- 
utes. In  dried  sputum  they  die  within  one  or  two  hours.  They  are 
easily  killed  even  by  the  weaker  antiseptics.  Upon  culture  media  the 
bacilli,  if  not  transplanted,  die  within  a  week  or  less,  the  time  depend- 
ing to  some  extent  upon  the  medium  used. 

Pathogenicity. — The  relationship  between  the  clinical  disease  known 
as  influenza  or  grippe  and  the  Pfeiffer  bacillus  has  been  definitely  estab- 
lished by  numerous  investigators.^  During  epidemics,  the  bacilli  are 
found  with  much  regularity  in  the  nasal  passages  and  bronchial  secre- 
tions of  those  afflicted  with  the  malady.  The  organs  most  frequently 
attacked  in  man  are  the  upper  respiratory  passages  and  lungs.  Here 
the  disease  most  frequently  takes  the  form  of  a  broncho-  or  lobular 
pneumonia,  and  sections  of  the  lung  tissue  of  those  who  have  died  of  the 
infection  show  innumerable  bacilli  upon  and  within  the  mucosa  of  the 
bronchioles.  [Thin  sections  are  stained  for  one-half  to  one  hour  in 
dilute  carbol  fuchsin  and  are  then  dehydrated  in  slightly  acid  alcohol 
(alcohol  absolute  §  i,  glacial  acetic  acid  gtt.  i-ij).] 

Clinically,  influenzal  broncho-pneumonias  are  not  essentially  dif- 
ferent from  those  due  to  other  microorganisms,  and  it  must  always 
be  left  to  the  bacteriological  examination  to  make  the  positive 
diagnosis.  Pulmonary  influenzal  infection  is  not  infrequently  followed 
by  abscess  or  gangrene  of  the  lung,  and  occasionally  develops  into  a 
chronic  interstitial  process.  The  bacilli  have  also  been  found  in  the 
middle  ear,^  in  the  meninges,^  and  in  the  brain  and  spinal  cord.  Bacilli 
in  the  circulating  blood  have  never  been  satisfactorily  demonstrated, 
although  the  general  characters  of  the  symptoms  would  suggest  a  septi- 
cemia. The  short  incubation  period  ^  of  the  disease  was  involuntarily 
determined  by  Kretz,  who  fell  ill  twenty-four  hours  after  accidentally 
breaking  an  agar  plate  of  a  pure  culture  which  he  was  photographing. 

The  bacilli  are  said  to  remain  in  the  bronchial  secretions  of  conval- 

^ Kruse,  in  Fltigge,  "Die  Mikroorg.,"  Leipzig,  1896. 

2  Weichselbaum,  Wien.  klin.  Woch.,  32,  1892;  Baumler,  Miinch.  med.  Woch., 
1894;  Huber,  loc.  cit. 

'  Kossell,  Charite-Annalen,  1893.  ■*  Pfuhl,  Berl.  klin.  Woch.,  xxxix,  1892. 

6  Quoted  from  Tedesco,  Cent.  f.  Bakt.,  xliii,  1907. 


BACILLUS  INFLUENZAE  641 

escents  or  even  of  normal  individuals  for  many  years.  They  are  found 
for  long  periods  in  the  lungs  of  those  suffering  from  tuberculosis.  To 
such  sources,  probably,  are  attributable  the  sporadic  cases  developing 
constantly  in  crowded  communities.  Occasional  reactivation  of  the 
influenzal  infection  may  often  aggravate  the  condition  of  phthisical 
patients.  Cases  of  influenza  observed  apart  from  the  large  epidemics 
are  rarely  due  to  an  unmixed  Pfeiffer  bacillus  infection,  but  are  usually 
due  to  a  mixed  infection,  including  with  this  bacillus,  pneumococci, 
streptococci,  and  other  secondary  invaders.^  This  may,  in  part,  account 
for  the  frequently  atypical  courses  of  such  attacks. 

Dr.  Anna  Williams  ^  has  recently  studied  hemoglobinophilic  bacilli 
isolated  from  the  eye  in  cases  of  trachoma.  She  believes  that  trachoma 
is  probably  caused  by  bacteria  of  this  group.  At  first  an  acute  infection 
or  acute  conjunctivitis  occurs.  Later  when  chronic  productive  inflam- 
mation supervenes  the  clinical  picture  is  that  of  trachoma. 

Experimental  infection  of  animals  reveals  susceptibihty  only  in 
monkeys.  Pfeiffer  and  Beck^  produced  influenza-like  symptoms  in  mon- 
keys by  rubbing  a  pure  culture  of  the  bacillus  upon  the  unbroken  nasal 
mucosa.  Intravenous  inoculation  in  rabbits  produced  severe  symptoms, 
but  the  bacilli  do  not  seem  to  proliferate  in  these  animals,  the  reaction 
probably  being  purely  toxic.  Cultures  killed  with  chloroform  may  pro- 
duce severe  transient  toxic  symptoms  in  rabbits.^  Immunity  produced 
by  an  attack  of  influenza,  if  present  at  all,  is  of  very  short  duration. 

Bacteria  Closely  Related  to  Influenza  Bacillus. — Pseudo-influenza 
Bacillus. — In  the  broncho-pneumonic  processes  of  children,  Pfeiffer  ^ 
found  small,  non-motile.  Gram-negative  bacilli,  which  he  was  forced  to 
separate  from  true  influenza  bacilli  because  of  their  slightly  greater  size, 
and  their  tendency  to  form  threads  and  involution  forms.  These  micro- 
organisms are  strictly  aerobic  and  grow,  like  true  influenza  bacilli,  only 
upon  blood  media.  They  are  differentiated  entirely  by  their  morphology 
upon  twenty-four-hour-old  blood-agar  cultures.  Wollstein,^  who  has 
made  a  careful  study  of  influenza-like  bacilli,  both  culturally  and  by 
agglutination  tests,  has  come  to  the  conclusion  that  these  bacilli  are  so 
similar  to  the  true  influenza  organisms  that  the  term  pseudo-influenza 
should  be  discarded.     Strains  of  similar  bacilli  isolated  from  cases  of 

1  Tedesco,  loc.  cit. 

2  Dr.  Anna  Williams,  Inter.  Congress  of  Hygiene  and  Demography,  Washington, 
1912.  3  Pfeiffer  und  Beck,  Deut.  med.  Woch.,  xxi,  1893. 

*  Pfeiffer,  loc.  cit.  ^  Pfeiffer,  Zeit.  f.  Hyg.,  xiii,  1892. 

« Wollsiein,  Jour.  Exp.  Med.,  viii,  1906. 


542 


PATHOGENIC  MICROORGANISMS 


pertussis,  while  differing  from  the  others  in  some  of  their  character- 
istics, could  not  properly  be  maintained  as  distinct  species. 

Koch-Weeks  Bacillus. — Koch;^  in  1883,  Weeks  ^  and  Kartulis,  in 
1887,  described  a  small  Gram-negative  bacillus  found  in  connection 
with  a  form  of  acute  conjunctivitis  which  occurs  epidemically.  The 
bacillus  is  morphologically  similar  to  B.  influenzae,  but  is  generally 
longer  than  this  and  more  slender.  The  bacilli  grow  only  at  incuba> 
tor  temperature.  Recent  studies  by  Anna  Williams  at  the  New  York 
Department  of  Health  seem  to  indicate  that  the  Koch-Weeks  bacillus 
may  be  merely  a  variety  of  the  true  influenza  bacillus. 

Bacillus  of  Pleuro-Pneumonia  of  Rabbits. — This  is  a  small 


Fig.  115. — Koch- Weeks  Bacillus. 


Gram-negative  bacillus,  described  by  Beck,  not  unlike  that  of  influenza. 
These  microorganisms  are  slightly  larger  than  the  Pfeiffer  bacilli  and 
grow  upon  ordinary  media  even  without  animal  sera  or  hemoglobin. 

Bacillus  murisepticus  and  Bacillus  RHUsioPATHiiE. — While  mor- 
phologically similar  to  the  microorganisms  of  this  group,  these  bacilli 
are  culturally  easily  separated  because  of  their  luxuriant  growth  on 
simple  media.  (The  last  two  microorganisms  are  more  closely  related 
to  the  groups  of  the  bacilli  of  hemorrhagic  septicemia.     See  page  561.) 

1  Koch,  Arb.  a.  d.  kais.  Gesundheitsamt,  iii;  Cent.  f.  Bakt.,  1,  1887. 

2  Weeks,  N.  Y.  Eye  and  Ear  Infirmary  Rep.,  1895;  Arch.  f.  Augenheilk.,  1887. 

3  Kamen,  Cent.  f.  Bakt.,  xxv,  1899;  Weichselbaum  and  Miiller,  Arch.  f.  Ophthalm., 
xlvii,  1899;  Knapp,  Studies  from  Dept.  of  Path.,  Coll.  of  P.  and  S.,  1903. 


CHAPTER  XXXVIII 

BORDET-GENGOU     BACILLUS,     MORAX-AXENFELD     BACILLUS,    ZUR 
NEDDEN'S  BACILLUS,  DUCREY  BACILLUS 

BORDET-GENGOU  BACILLUS 

{"  Microbe  de  la  Coqueluche,''  Pertussis  hacUlus,  Bacilliis  of  whooping- 
cough.) 

In  1900  Bordet  and  Gengou  ^  observed  in  the  sputum  of  a  child 
suffering  from  pertussis  a  small  ovoid  bacillus  which,  though  similar  to 
the  influenza  bacillus,  showed  a  number  of  morphological  characteristics 
which  led  them  to  regard  it  as  a  distinct  species.  As  they  were  at  first 
unable  to  cultivate  this  organism,  their  discovery  remained  ques- 
tionable until  1906,  when  cultivation  succeeded  and  the  biology  of  the 
microorganism  was  more  fully  elucidated. 

Morphology. — The  morphology  of  this  organism  is  described  by 
them  as  follows:  The  organism  in  the  sputum,  early  in  the  disease,  is 
scattered  in  enormous  numbers  indiscriminately  among  the  pus  cells, 
and  at  times  within  the  cells.  It  is  extremely  small  and  ovoid,  and 
frequently  is  so  short  that  it  resembles  a  micrococcus.  Often  its  poles 
stain  more  deeply  than  the  center.  In  general,  the  form  of  the  or- 
ganisms is  constant,  though  occasionally  slightly  larger  individuals 
are  encountered.  -They  are  usually  grouped  separately,  though  occa- 
sionally in  pairs,  end  to  end. 

Compared  with  the  influenza  bacillus  in  morphology,  the  bacillus 
of  pertussis  is  more  regularly  ovoid  and  somewhat  larger.  It  has, 
furthermore,  less  tendency  to  pleomorphism  and  involution. 

Staining. — The  Bordet-Gengou  bacillus  may  be  stained  with  alkaline 
methylene-blue,  dilute  carbol-fuchsin,  or  aqueous  fuchsin  solutions. 
Bordet  and  Gengou  recommended  as  a  staining-solution  carbolated 
toluidin-blue  made  up  as  follows: 

Toluidin-blue 5  gms. 

Alcohol 100  c.c. 

Water 500  c.c. 

1  Bordet  et  Gengou,  Ann.  de  I'inst.  Pasteur,  1906. 
36  543 


544  PATHOGENIC  MICROORGANISMS 

Allow  to  dissolve  and  add  500  c.c.  of  5  per  cent  carbolic  acid  in  water.  Let  this 
stand  one  or  two  days  and  filter. 

Stained  by  the  method  of  Gram,  the  bacillus  of  Bordet  and  Gengou 
is  decolorized. 

Cultivation. — Early  attempts  at  cultivation  made  by  the  discov- 
erers upon  ©rdinary  ascitic  agar  or  blood  agar  were  unsuccessful.  They 
finally  obtained  successful  cultures  from  sputum  by  the  use  of  the 
following  medium: 

One  hundred  grams  of  shced  potato  are  put  into  200  c.c.  of  4  per 
cent  glycerin  in  water.  This  is  steamed  in  an  autoclave  and  a  glycerin 
extract  of  potato  obtained.  To  50  c.c.  of  this  extract  150  c.c.  of  6-per- 
cent salt  solution  and  5  grams  of  agar  are  added.  The  mixture  is  melted 
in  the  autoclave  and  the  fluid  filled  into  test  tubes,  2  to  3  c.c.  each,  and 

sterilized.  To  each  tube,  after  sterilization, 
is  added  an  equal  volume  of  sterile  de- 
fibrinated  rabbit  blood  or  preferably  human 
blood,  the  substances  are  mixed,  and  the 
tubes  slanted. 

On  such  a  medium,  inoculated  with 
sputum,  taken  preferably  during  the  par- 
oxysms of  the  first  day  of  the  disease, 
colonies  appear,  which  are  barely  visible 
after  twenty-four  hours,^  plainly  visible 
Fig.  116.-Bordet-Gengou  ^^^^^  forty-eight  hours.  They  are  small. 
Bacillus.  grayish,  and  rather  thick.     After  the  first 

generation  the  organisms  grow  with  mark- 
edly greater  luxuriance  and  speed.  On  the  potato-blood  medium, 
after  several  generations  of  artificial  cultivation,  they  form  a  grayish 
glistening  layer  which,  after  a  few  days,  becomes  heavy  and  thick, 
almost  resembling  the  growth  of  typhoid  bacilli.  In  these  later 
generations,  also,  they  develop  readily  upon  plain  blood  agar  or 
ascitic  agar  and  in  ascitic  broth  or  broth  to  which  blood  has  been 
added.  In  the  fluid  media  they  form  a  viscid  sediment,  but  no  pellicle. 
Culturally,  the  'bacillus  varies  from  B.  influenzae  in  growing  less 
readily  on  hemoglobin  media  than  the  latter,  on  first  cultivation  from  the 
sputum.  Later  it  grows  much  more  heavily  on  such  media  and  shows 
less  dependence  upon  the  presence  of  hemoglobin  than  does  B.  influenzae. 
It  also  grows  rather  more  slowly  than  the  influenza  bacillus.      It  is 

1  Wollstein,  Jour.  Exp.  Med.,  xi,  1909. 


w  ",^"' 

-  •  "     1? 

.  '*'*   '* 

^^.' 

^^■,-'- 

U: 

.c  i?' •' 

«.»**      • 

'  '^•■ 

'     "    «^ 

--^•x 

1 

'-      ' 

*••     « 

BORDET-GENGOU  BACILLUS  545 

strictly  aerobic  and'  in  fluid  cultures  is  best  grown  in  wide  flasks  with 
shallow  layers  of  the  medium. 

The  Bordet-Gengou  bacillus  grows  moderately  at  temperatures  about 
37.5°  C,  but  does  not  cease  to  grow  at  temperatures  as  low  as  5°  to  10°  C. 
On  blood  agar  and  in  ascitic  broth  it  may  remain  alive  for  as  long  as  two 
months  (Wollstein). 

Pathogenicity. — As  regards  the  pathogenicity  and  etiological  spe- 
cificity of  this  organism  for  whooping-cough,  no  positive  statement 
can  as  yet  be  made.  The  fact  that  it  has  been  found  in  many  cases  in 
almost  pure  cultures  during  the  early  paroxysms,  renders  it  likely  that 
the  organism  is  the  specific  cause  of  the  disease.  However,  in  early 
cases  true  influenza  bacilli  have  been  often  found,  and  these  latter  seem 
to  remain  in  the  sputum  of  such  patients  for  a  longer  period  and  in 
larger  numbers  than  the  bacillus  of  Bordet  and  Gengou.  Endotoxins 
have  been  obtained  from  the  cultures  of  the  bacilli  by  Bordet  and  Gengou 
by  the  method  of  Besredka.^  The  growth  from  slant  cultures  is  washed 
up  in  a  little  salt  solution,  dried  in  vacuo,  and  ground  in  a  mortar  with 
a  small,  measured  quantity  of  salt.  Finally,  enough  distilled  water  is 
added  to  bring  the  salt  into  a  solution  of  0.75  per  cent  and  the  mixture 
is  centrifugalized  and  decanted.  One  to  two  c.c.  of  such  an  extract  will 
usually  kill  a  rabbit  within  twenty-four  hours  after  intravenous  inocula- 
tion. Subcutaneous  inoculation  produces  non-suppurating  necrosis  and 
ulceration  without  marked  constitutional  symptoms. 

Inoculation  of  monkeys  with  the  bacilli  themselves  by  the  respira- 
tory path  has  failed  to  produce  the  disease. 

Specific  agglutinins  may  be  obtained  in  immunized  animals  which 
prove  absolutely  the  distinctness  of  this  organism  from  Bacillus  in- 
fluenzae.^  In  the  serum  of  afflicted  children  the  agglutination  is  too 
irregular  to  be  of  value. 

Specific  complement  fixation  with  the  serum  of  patients  is  reported 
by  Bordet  and  Gengou,  but  failed  in  the  hands  of  Wollstein. 

MORAX-AXENFELD  BACILLUS 

In  1896  Morax^  described  a  diplo-bacillus,  which  he  associated 
etiologically  with  a  type  of  chronic  conjunctivitis  to  which  he  applied 
the  name  "  conjonctivite  suhaigueJ^  Soon  after  this,  a  similar  micro- 
organism was  found  in  cases  corresponding  to  those  of  Morax  by  Axen- 

1  Bordet,  Bull,  de  la  Soc.  Roy.  de  Brux.,  1907. 
^  Wollstein,  loc.  cit.  ^  Morax,  Ann.  de  I'inst.  Pasteur,  1896. 


546  PATHOGENIC  MICROORGANISMS 

feld.i  The  condition  which  these  microorganisms  characteristically 
produce  is  a  catarrhal  conjunctivitis  which  usually  attacks  both  eyes. 
The  inflammation  is  especially  noticeable  in  the  angles  of  the  eye,  most 
severe  at  or  about  the  caruncle.  There  is  rarely  much  swelling  of  the 
conjunctiva  and  hardly  ever  ulceration.  The  condition  runs  a  subacute 
or  chronic  course.  Its  diagnosis  is  easily  made  by  smear  preparations 
of  the  pus  which  is  formed  with  especial  abundance  during  the  night. 

Morphology. — In  smear  preparations  from  the  pus,  the  microorgan- 
isms appear  as  short,  thick  bacilli,  usually  in  the  form  of  two  placed 
end  to  end,  but  not  infrequently  singly  or  in  short  chains.    Their  ends 


Fig.  117. — Morax-Axenfeld  Diplo-Bacillxjs. 

are  distinctly  rounded,  their  centers  slightly  bulging,  giving  the  bacillus 
an  ovoid  form.    They  are  usually  about  two  micra  in  length. 

They  are  easily  stained  by  the  usual  anilin  dyes,  and,  stained  by 
the  method  of  Gram,  are  completely  decolorized. 

Cultivation. — The  Morax-Axenfeld  bacillus  can  be  cultivated  only 
upon  alkaline  media  containing  blood  or  blood  serum. 

It  grows  poorly,  or  not  at  all,  at  room  temperature. 

Upon  Loeffler^s  blood  serum,  colonies  appear  after  twenty-four  to 
thirty-six  hours  as  small  indentations  which  indicate  a  liquefaction  of 
the  medium.    Axenfeld  states  that  eventually  the  entire  medium  may 

1  Axenfeldy  Cent,  f .  Bakt.,  xxi,  1897. 


ZUR  NEDDEN'S  BACILLUS  547 

become  liquefied.  Upon  serum  agar  delicate  grayish  drop-like  colonies 
are  formed  which  are  not  unlike  those  of  the  gonococcus. 

In  ascitic  bouillon  general  clouding  occurs  within  twenty-four  hours. 

Pathogenicity. —  Attempts  to  produce  lesions  in  the  lower  animals 
with  this  bacillus  have  been  universally  unsuccessful.  Subacute  con- 
junctivitis, however,  has  been  produced  in  human  beings  by  inocula- 
tions with  pure  cultures. 

ZUR  NEDDEN'S  BACILLUS 

In  ulcerative  conditions  of  the  cornea,  Zur  Nedden  has  frequently 
found  a  bacillus  to  which  he  attributes  etiological  importance. 

The  bacillus  which  he  has  described  is  small,  usually  less  than 
one  micron  in  length,  often  slightly  curved,  and  generally  found  singly. 
It  may  be  found  in  the  diplo  form  but  does  not  form  chains.  It  is 
stained  by  the  usual  dyes,  often  staining  poorly  at  the  ends.  Stained 
by  Gram's  method  it  is  decolorized.    The  bacillus  is  non-motile. 

Cultivation. — It  is  easily  cultivated  upon  the  ordinary  laboratory 
media.  Upon  agar  it  forms,  within  twenty-four  hours,  transparent, 
slightly  fluorescent  colonies  which  are  round,  raised,  rather  coarsely 
granular,  and  show  a  tendency  to  confluence. 

Gelatin  is  not  liquefied. 

Milk  is  coagulated. 

Upon  potato,  there  is  a  thick  yellowish  growth. 

Upon  dextrose  media,  there  is  acid  formation,  but  no  gas. 

The  bacillus  forms  no  indol  in  pepton  solutions. 

Pathogenicity. — Corneal  ulcers  have  been  produced  by  inoculation 
of  guinea-pigs. 

BACILLUS  OP  DUCREY 

The  soft  chancre,  or  chancroid,  is  an  acute  inflammatory,  destructive 
lesion  which  occurs  usually  upon  the  genitals  or  the  skin  surrounding 
the  genitals.  The  infection  is  conveyed  from  one  individual  to  an- 
other by  direct  contact.  It  may,  however,  under  conditions  of  surgical 
manipulation,  be  transmitted  indirectly  by  means  of  dressings,  towels, 
or  instruments. 

The  lesion  begins  usually  as  a  small  pustule  which  rapidly  ruptures, 
leaving  an  irregular  ulcer  with  undermined  edges  and  a  necrotic  floor 
which  spreads  rapidly.  It  differs  cHnically  from  the  true  or  syphilitic 
chancre  in  the  lack  of  induration  and  in  its  violent  inflammatory 


548  PATHOGENIC  MICROORGANISMS 

nature.  Usually  it  leads  to  lymphatic  swellings  in  the  groin  which, 
later,  give  rise  to  abscesses,  commonly  spoken  of  as  "buboes." 

In  the  discharges  from  such  lesions,  Ducrey,^  in  1889,  was  able  to 
demonstrate  minute  bacilli  to  which  he  attributed  an  etiological  rela- 
tionship to  the  disease,  both  because  of  the  regularity  of  their  presence 
in  the  lesions  and  the  successful  transference  of  the  disease  by  means 
of  pus  containing  the  microorganisms. 

Morphology  and  Staining. — The  Ducrey  bacillus  is  an  extremely 
small  bacillus,  measuring  from  one  to  two  micra  in  length  and  about 
half  a  micron  in  thickness.  It  has  a  tendency  to  appear  in  short 
chains  and  in  parallel  rows,  but  many  of  the  microorganisms  may  be 
seen  irregularly  grouped.  It  is  not  motile,  possesses  no  flagella,  and 
does  not  form  spores. 

Stained  by  the  ordinary  anilin  dyes,  it  has  a  tendency  to  take  the 
color  irregularly  and  to  appear  more  deeply  stained  at  the  poles.  By  the 
Gram  method,  it  is  decolorized.  In  tissue  sections,  it  may  be  demon- 
striited  by  Loeffler's  methylene-blue  method,  and  in  such  preparations 
has  been  found  within  the  granulation  tissues  forming  the  floor  of  the 
ulcers.    In  pus,  the  bacilli  are  often  found  within  leucocytes. 

Cultivation  and  Isolation. — Early  attempts  at  cultivation  of  this 
bacillus  were  universally  unsuccessful  in  spite  of  painstaking  experi- 
ments with  me^ia  prepared  of  human  skin  and  blood  serum.  In  1900, 
Besangon,  Griffon,  and  Le  Sourd  ^  finally  succeeded  in  obtaining  growths 
upon  a  medium  containing  agar  to  which  human  blood  had  been  added. 
They  were  equally  successful  when  dog's  or  rabbit's  blood  was  substi- 
tuted for  that  of  man.  Since  the  work  by  these-  authors,  the  cultiva- 
tion by  similar  methods  has  been  carried  out  by  a  number  of  investiga- 
tors. Coagulated  blood,  which  has  been  kept  for  several  days  in  sterile 
tubes,  has  been  found  to  constitute  a  favorable  medium.  Freshly 
clotted  blood  can  not  be  employed,  probably  because  of  the  bacteri- 
cidal action  of  the  serum.  Serum-agar  has  occasionally  been  used 
with  success,  but  does  not  give  results  as  satisfactory  as  those  obtained 
by  the  use  of  the  whole  blood. 

The  best  method  of  obtaining  pure  cultures  upon  such  media  con- 
sists in  puncturing  an  unruptured  bubo  with  a  sterile  hypodermic  needle 
and  transferring  the  pus  in  considerable  quantity  directly  to  the  agar. 
If  possible,  the  inoculation  of  the  media  should  be  made  immediately 

1  Ducrey,  Monatschr.  f.  prakt.  Dermat.,  9^  1889. 

2  Besangon,  Griffon,  et  Le  Sourd,  Presse  m^d.,  1900. 


BACILLUS  OF  DUCREY  549 

before  the  pus  has  had  a  chance  to  cool  off  or  to  be  exposed  to  light. 
When  buboes  are  not  available,  the  primary  lesion  may  be  thoroughly 
cleansed  with  sterile  water  or  salt  solution,  and  material  scraped  from 
the  bottom  of  the  ulcer  or  from  beneath  its  overhanging  edges  with  a 
stiff  platinum  loop.  This  material  is  then  smeared  over  the  surface  of  a 
number  of  blood-agar  plates. 

Upon  such  plates,  isolated  colonies  appear,  usually  after  forty-eight 
hours.  They  are  small,  transparent,  and  gray,  and  have  a  rather  firm, 
finely  granular  consistency.  The  colonies  rarely  grow  larger  than  pin- 
head  size,  and  have  no  tendency  to  coalesce.  At  room  temperature, 
the  cultures  die  out  rapidly.  Kept  in  the  incubator,  however,  they  may 
remain  alive  and  virulent  for  a  week  or  more. 

On  the  simpler  media,  glucose-agar,  broth,  or  gelatin,  cultivation 
is  never  successful.  On  moist  blood-agar  and  in  the  condensation 
water  of  such  tubes,  the  bacilli  have  a  tendency  to  grow  out  in  long 
chains.  Upon  media  which  are  very  dry,  they  appear  singly  or  in 
short  chains. 

Pathogenicity. — Besangon,  Griffon,  and  Le  Sourd,  and  others,  have 
succeeded  in  producing  lesions  in  man  by  inoculation  with  pure  cultures. 
Inoculation  of  the  lower  animals  has,  so  far,  been  entirely  without  result. 

MICROCOCCUS  MELITENSIS  (MALTA  FEVER) 

(Bacillus  melitensis) 

Malta  fever  is  a  disease  occurring  along  the  Mediterranean  coast 
and  its  islands.  It  has  been  recently  found  to "  occur  also  in  South 
America,  South  Africa,  China,  and  in  the  West  Indies.  The  disease 
is  not  very  unlike  typhoid  fever,  though  more  irregular  and  with  a 
lower  mortality.  It  is  accompanied  by  joint  pains,  sweating,  constipa- 
tion, and  occasionally  orchitis.     The  spleen  is  almost  always  enlarged. 

Recent  investigations  into  the  manner  in  which  this  disease  is  con- 
veyed have  revealed  that  it  is  primarily  an  infection  of  goats.  A  large 
percentage  of  the  goats  on  Malta  were  shown  to  be  infected  and  passed 
the  organism  with  the  milk.  Forty  per  cent  of  the  goats  gave  positive 
agglutination  tests  and  the  organisms  have  been  found  in  the  milk  in 
about  10  per  cent  of  the  animals. 

The  most  susceptible  animals  seem  to  be  goats,  but  horses  and  cows 
are  also  susceptible.  In  guinea-pigs  and  rabbits  the  disease  can  be  ex- 
perimentally produced,  but  usually  takes  a  protracted  course.    Monkeys 


550  PATHOGENIC  MICROORGANISMS 

are  susceptible,  and  the  disease  produced  in  these  animals  is  in  many- 
features  identical  with  that  of  man. 

Transmission  seems  to  take  place  chiefly  by  the  ingestion  of  infected 
milk.  Direct  cutaneous  infection  or  through  mucous  membranes  may 
also  occur.  In  human  beings,  suffering  from  the  disease,  the  organisms 
may  be  isolated  from  the  blood  stream  during  the  entire  course  of 
the  disease  and  as  early  as  the  second  day.  The  disease  is  rarely 
fatal,  death  occurring  in  less  than  2  per  cent  of  the  cases  (Eyre,  loc.  cit.)} 

The  microorganism  causing  the  disease  was  isolated  in  1887  by 
Bruce,^  a  British  army  surgeon. 

Morphology. — Micrococcus  melitensis  is  a  minute  bacterium  ap- 
pearing coccoid  in  smears  from  agar  cultures,  in  broth  cultures  assum- 
ing the  form  of  a  short,  slightly  wedge-shaped  bacillus  resembling  B. 
influenzae.  Babes  ^  regards  it  as  unquestionably  a  bacillus.  Eyre  de- 
scribes it  as  a  minute  coccus,  and  beUeves  the  bacillus-hke  individuals 
to  represent  involution  forms.  It  appears  in  irregularly  parallel  groups, 
and  occasionally  forms  short  chains. 

It  is  easily  stained  with  the  ordinary  dyes,  and  is  decolorized  by 
Gram's  method. 

Cultivation. — Micrococcus  melitensis  can  usually  be  cultivated  from 
the  spleens  of  those  who  have  succumbed  to  the  disease  and  from  the 
blood  stream  in  active  cases.  It  grows  on  nutrient  agar  at  37.5°  C, 
forming  small,  pearly  white  colonies  at  the  end  of  two  or  three  days. 
It  grows  easily  on  all  of  the  ordinary  laboratory  media. 

Both  in  patients  and  in  injected  animals,  infection  with  this  bacte- 
rium produces  specific  agglutinins  which  are  of  great  practical  aid  in 
diagnosis.^ 

1  British  Commission  Report  cited  from  Eyre  in  Kolle  und  Wassermanrit 
Handbuch,  etc.,  Erganzungsband,  Heft  2. 

2  Bruce,  Practitioner,  1887. 

'  Babes,  Kolle  und  Wassermann,  iii,  p.  443. 

*  Wright  and  Lamb,  Jour.  Path,  and  Bact.,  v,  1899. 


CHAPTER  XXXIX 

THE    BACILLI  OF   THE   HEMORRHAGIC    SEPTICEMIA   GROUP 
AND    BACILLUS    PESTIS. 

In  many  of  the  lower  animals  there  occur  violently  acute  bacterial 
infections  characterized  by  general  septicemia,  usually  with  petechial 
hemorrhages  throughout  the  organs  and  serous  membranes  and  severe 
intestinal  inflammations.  These  diseases,  spoken  of  as  the  "hemor- 
rhagic septicemias,''  are  caused  by  a  group  of  closely  allied  bacilli,  first 
classified  together  by  Hueppe  ^  in  1886.  Some  confusion  has  existed 
as  to  the  forms  which  should  be  considered  within  Hueppe's  group  of 
"hemorrhagic  septicemia,"  a  number  of  bacteriologists  including  in 
this  class  bacilli  such  as  Loefl^er's  Bacillus  typhi  murium,  and  Salmon 
and  Smith's  hog-cholera  bacillus,  microorganisms  which,  because  of 
their  motility  and  cultural  characteristics,  belong  more  properly  to  the 
"Gartner,"  " enteritidis,"  or  "paratyphoid"  group,  intermediate  be- 
tween colon  and  typhoid. 

The  organisms  properly  belonging  to  this  group  are  short  bacilli,  more 
plump  than  are  those  of  the  colon  type,  and  showing  a  marked  ten- 
dency to  stain  more  deeply  at  the  poles  than  at  the  center.  They  are 
non-motile,  possess  no  flagella,  and  do  not  form  spores.  They  grow 
readily  upon  simple  media,  but  show  a  very  marked  preference  for 
oxygen,  growing  but  slightly  below  the  surface  of  media.  By  some 
observers  they  are  characterized  as  "obligatory  aerobes,"  but  this  is 
undoubtedly  a  mistake. 

\Miile  showing  considerable  variations  in  form  and  differences  in 
minor  cultural  characteristics,  the  species  characteristics  of  polar  stain- 
ing, decolorization  by  Gram,  immobility,  lack  of  gelatin  liquefaction, 
and  great  pathogenicity  for  animals,  stamp  alike  all  members  of  the 
group.  Its  chief  recognized  representatives  are  the  bacillus  of  chicken 
cholera,    the    bacillus    of    swine-plague    (Deutsche    Schweineseuche) , 

» Hueppe,  Berl.  klin.  Woch.,  1886. 
551 


552  PATHOGENIC  MICROORGANISMS 

and  the   Bacillus  pleurosepticus  which  causes    an   acute    disease  in 
cattle  and  often  in  wild  game. 

Because  of  certain  cultural  and  pathogenic  characteristics,  it 
seems  best  to  consider  the  bacillus  of  bubonic  plague  with  this  group. 

BACILLUS   OF   CHICKEN   CHOLERA 

(Bacilltcs  avisepticus) 

The  bacillus  of  chicken  cholera  was  first  carefully  studied  by  Pas- 
teur ^  in  1880.  It  is  a  short,  non-motile  bacillus,  measuring  from  0.5  to 
1  micron  in  length.  Stained  with  the  ordinary  anilin  dyes,  it  displays 
marked  polar  staining  qualities,  which  often  give  it  the  appearance  of 
being  a  diplococcus.  It  is  decolorized  by  Gram's  method.  It  does  not 
form  spores,  but  may  occasionally  form  vacuolated  degeneration  forms, 
not  unlike  those  described  for  Bacillus  pestis. 

The  bacillus  is  easily  cultivated  from  the  blood  and  organs  of  infected 
animals,  it  grows  well  upon  the  simplest  media  at  temperatures  vary- 
ing from  25°  to  40°  C.  In  broth ,  it  produces  uniform  clouding  with 
later  a  formation  of  a  pellicle.  Upon  agar  it  forms,  within  twenty-four 
to  forty-eight  hours,  minute  colonies,  white  or  yellowish  in  color,  which 
are  at  first  transparent,  later  opaque.  Upon  gelatin^  it  grows  without 
liquefaction.  Upon  milk,  the  growth  is  slow  and  does  not  produce  co- 
agulation. According  to  Kruse,^  indol  is  formed  from  pepton  bouillon. 
Acid,  but  no  gas,  is  formed  in  sugar  broth. 

Among  barnyard  fowl,  this  disease  is  widely  prevalent,  attacking 
chickens,  ducks,  geese,  and  a  large  variety  of  smaller  birds.  The  infection 
is  extremely  acute,  ending  fatally  within  a  few  days.  It  is  accompanied 
by  diarrhea,  often  with  bloody  stools,  great  exhaustion,  and,  toward  the 
end,  a  drowsiness  bordering  on  coma.  Autopsy  upon  the  animals  re- 
veals hemorrhagic  inflammation  of  the  intestinal  mucosa,  enlargement 
of  the  liver  and  spleen,  and  often  bronchopneumonia. 

The  specific  bacilli  may  be  found  in  the  blood,  in  the  organs,  in  exu- 
dates, if  these  are  present,  and  in  large  numbers  in  the  dejecta.  Infection 
takes  place  probably  through  the  food  and  water  contaminated  by  the 
discharges  of  diseased  birds.^ 

Subcutaneous  inoculation  or  feeding  of  such  animals  with  pure 
cultures,  even  in  minute  doses,  gives  rise  to  a  quickly  developing 
septicemia  which  is  uniformly  fatal.     The  bacillus  is  extremely  patho- 

»  Pasteur,  Comptes  rend,  de  I'acad.  des  sci.,  1880. 

siiTn^e,  in  Fliigge's  "Die  Mikroorganismen." 

3  Salmon,  Rep.  of  the  Com.  of  Agriculture,  1880,  1881,  and  1882. 


BACILLI   OF  HEMORRHAGIC   SEPTICEMIA  GROUP  553 

genie  for  rabbits,  less  so  for  hogs,  sheep,  and  horses,  if  infection  is  prac- 
ticed by  subcutaneous  inoculation.  Infection  by  ingestion  does  not 
seem  to  cause  disease  in  these  animals. 

Historically,  the  bacillus  of  chicken  cholera  is  extremely  interesting, 
since  it  was  with  this  microorganism  that  Pasteur  ^  carried  out  some  of 
his  fundamental  researches  upon  immunity,  and  succeeded  in  immu- 
nizing chickens  with  attenuated  cultures.  The  first  attenuation  ex- 
periment made  by  Pasteur  consisted  in  allowing  the  bacilli  to  remain  in 
a  broth  culture  for  a  prolonged  period  without  transplantation.  With 
minute  doses  of-  such  a  culture  (vaccin  I)  he  inoculated  chickens,  fol- 
lowing this,  after  ten  days,  with  a  small  dose  of  a  fully  virulent  culture. 
Although  enormously  important  in  principle,  the  practical  results  from 
this  method,  as  applied  to  chicken  cholera,  have  not  been  satisfactory. 
It  was  with  this  bacillus,  furthermore,  that  Pasteur  was  first  able  to 
demonstrate  the  existence  of  a  free  toxin  which  could  be  separated 
from  the  bacteria  by  filtration. 

BACILLUS   OP   SWINE   PLAGUE 

{Bacillus  suisepticuSj  Schweineseuche) 

This  microorganism  is  almost  identical  in  form  and  cultural  charac- 
teristics with  the  bacillus  of  chicken  cholera.  It  is  non-motile,  forms 
no  spores,  is  Gram-negative,  and  does  not  liquefy  gelatin.  The  bacillus 
causes  an  epidemic  disease  among  hogs,  which  is  characterized  almost 
regularly  by  a  bronchopneumonia  followed  by  general  septicemia. 
There  is  often  a  sero-sanguineous  pleural  exudate,  a  swelling  of  bronchial 
lymph  glands  and  of  liver  and  spleen.  The  gastrointestinal  tract  is 
rarely  affected.  The  bacilli  at  autopsy  may  be  found  in  the  lungs, 
in  the  exudates,  in  the  liver  and  spleen,  and  in  the  blood.  The  disease 
is  rarely  acute,  but,  in  young  pigs,  almost  uniformly  fatal. 

It  is  probable  that  spontaneous  infection  usually  occurs  by  inhala- 
tion. Experimental  inoculation  is  successful  in  pigs,  both  when  given 
subcutaneously  and  when  administered  by  the  inhalation  method. 
Mice,  guinea-pigs,  and  rabbits  are  also  susceptible,  dying  within  three 
or  four  days  after  subcutaneous  inoculation  of  small  doses. 

Active  and  passive  immunization  of  animals  against  Bacillus  suisep- 
ticus  has  been  attempted  by  various  observers.  Active  immunization, 
if  carried  out  with  care,  may  be  successfully  done  in  the  laboratory. 

1  Pasteur,  loc.  cit. 


554  PATHOGENIC  MICROORGANISMS 

Passive  immunization  of  animals  with  the  serum  of  actively  immunized 
horses  has  been  practiced  by  Kitt  and  Mayr/  Schreiber,^  and  Wasser- 
mann  and  Ostertag.  The  last-named  observers,  working  with  a  poly- 
valent serum  produced  with  a  number  of  different  strains  of  the  bacillus, 
have  obtained  results  of  considerable  practical  value.  The  researches 
of  Kitt  and  Mayr  have  revealed  a  fact  pointing  to  the  interrelationship 
of  the  bacilli  of  the  "hemorrhagic  septicemia"  group.  They  were  able 
to  show  that  the  serum  of  horses  immunized  with  chicken  cholera 
bacilli  was  able  to  protect,  somewhat,  against  Bacillus  suisepticus. 

Infection  with  the  bacillus  of  swine  plague,  in  hogs,  is  often  ac- 
companied by  an  infection  with  the  hog-cholera  bacilhis  (Schweinepest). 
The  latter,  as  we  have  seen,  is  a  microorganism  belonging  to  the  enteri- 
tidis  group,  intermediate  between  Bacillus  coli  and  Bacillus  typhosus,  and 
differing  from  suisepticus  in  being  actively  motile,  possessing  flagella, 
not  showing  the  polar  staining,  having  a  more  slender  morphology,  and 
producing  gas  upon  dextrose  broth.  A  confusion  between  the  two 
bacilli  frequently  occurs  because  of  their  nomenclature.  Bacteriologic- 
ally  and  pathogenically,  they  are  quite  distinct.  Bacillus  suisepticus 
produces  an  acute  septicemia,  accompanied  by  bronchopneumonia  and 
usually  not  affecting  the  gastro-intestinal  canal.  The  bacillus  of  hog 
cholera  produces  an  infection  localized  in  the  intestinal  canal. 

BACILLUS   PESTIS 

(Bacillus  of  Bubonic  Plague) 

The  history  of  epidemic  diseases  has  no  more  terrifying  chapter 
than  that  of  plague.^  Sweeping,  time  and  again,  over  large  areas  of 
the  civilized  world,  its  scope  and  mortality  were  often  so  great  that 
all  forms  of  human  activity  were  temporarily  paralyzed.  In  the 
reign  of  Justinian  almost  fifty  per  cent  of  the  entire  population  of 
the  Roman  Empire  perished  from  the  disease.  The  "Black  Death" 
which  swept  over  Europe  during  the  fourteenth  century  killed  about 
twenty-five  million  people.  Smaller  epidemics,  appearing  in  numerous 
parts  of  the  world  during  the  sixteenth,  seventeenth,  and  eighteenth 
centuries,  hare  claimed  innumerable  victims.  In  1893,  plague  appeared 
in  Hong  Kong.  During  the  epidemic  which  followed,  Bacillus  pestis, 
now  recognized  as  the  etiological  factor  of  the  disease,  was  discovered  by 

»  Kitt  und  Mayr,  Monatsh.  f.  prakt.  Thierheilk.,  8,  1897. 

^Schreiber,  Berl.  tierarztl.  Woch.,  10,  1899. 

«  Hirsch.  "  Handb.  d.  histor.-geogr.  Path.,"  1881. 


BACILLUS    PESTLS  555 

Kitasato  *  and  by  Yersin,^  independently  of  each  other.  By  both  ob- 
servers the  bacillus  could  invariably  be  found  in  the  pus  from  the  buboes 
of  afflicted  persons.  It  could  be  demonstrated  in  enormous  numbers 
in  the  cadavers  of  victims.  The  constancy  of  the  occurrence  of  the 
bacillus  in  patients,  shown  in  the  innumerable  researches  of  many 
bacteriologists,  would  alone  be  sufficient  evidence  of  its  etiological 
relationship  to  the  disease.  This  evidence  is  strengthened,  moreover, 
by  accidental  infections  which  occurred  in  Vienna  in  1898,  with  labora- 
tory cultures. 

Morphology  and  Staining.— Bacillus  pestis  is  a  short,  thick  bacillus 
with  well-rounded  ends.    Its  length  is  barely  two  or  two  and  a  half  times 


Fig.  118. — Bacillus  pestis.    (After  Mallory  and  Wright.) 

its  breadth  (1.5  to  1.75  micra  by  0.5  to  0.7  micron).  The  bacilli  appear 
singly,  in  pairs,  or,  more  rarely,  iii  short  chains  of  three  or  more.  They 
show  distinct  polar  staining.  In  size  and  shape  these  bacilli  are  sub- 
ject to  a  greater  degree  of  variation  than  are  most  other  microorganisms. 
In  old  lesions  or  in  old  cultures  the  bacilli  show  involution  forms  which 
may  appear  either  as  swollen  coccoid  forms  or  as  longer,  club-shaped, 
diphtheroid  bacilli.  Degenerating  individuals  appear  often  as  swollen, 
oval  vacuoles.  All  these  involution  forms,  by  their  very  irregularity, 
a?e  of  diagnostic  importance.  They  appear  more  numerous  in  artificial 
fiultures  than  in  human  lesions. 

According   to  Albrecht  and  Ghon,^  the  plague    bacillus  may,  by 

» Kitasato,  Lancet,  1894.  2  Yersin,  Ann.  de  I'inst.  Pasteur,  1894. 

»  Albrecht  und  Ghon,  Wien,  1898. 


556  PATHOGENIC  MICROORGANISMS 

special  methods,  be  shown  to  possess  a  gelatinous  capsule.     It  does 
not  possess  flagella  and  does  not  form  spores. 

The  plague  bacillus  is  easily  stained  with  all  the  usual  anilin 
dyes.  Diluted  aqueous  fuchsin  and  methylene-blue  are  most  frequently 
employed.  With  these  stains  the  characteristically  deeper  staining 
of  the  polar  portions  of  the  bacillus  is  usually  easy  to  demonstrate. 
Special  polar  stains  have  been  devised  by  various  observers.  Most  of 
these  depend  upon  avoidance  of  the  usual  heat  fixation  of  the  prepara- 
tions, which,  in  some  way,  seems  to  interfere  with  good  polar  staining. 
Fixation  of  the  dried  smears  with  absolute  alcohol  is.  therefore,  prefer- 
able.   The  bacillus  is  decolorized  by  Gram's  method. 


Fig.  119. — Bacillus  pestis.  Involution  Forms.     (After  Zettnow.) 

Isolation  and  Cultivation. — The  bacillus  is  easily  isolated  in  pure 
culture  from  the  specific  lesions  of  plague  patients,  during  life  or  at 
autopsy.  It  grows  readily  and  luxuriantly  upon  the  meat-infusion 
media.  The  optimum  temperature  for  its  cultivation  is  about  30°  C. 
Below  20°  C.  and  above  38°  C,  growth  is  sparse  and  delayed,  though  it 
is  not  entirely  inhibited  until  exposed  to  temperatures  below  12°  C, 
or  above  40°  C.  The  most  favorable  reaction  of  culture  media  is  neu- 
trality or  moderate  alkalinity,  though  slight  acidity  does  not  prevent 
development. 

On  agar,  growth  appears  within  twenty-four  hours  as  minute 
colonies  with  a  compact  small  center  surrounded  by  a  broad,  irregularly 
indented,  granular  margin. 


BACILLUS  PESTIS  557 

On  gelatin,  similar  colonies  appear  after  two  or  three  days  at  20° 
to  22°  C.     The  gelatin  is  not  liquefied. 

In  bouillon,  the  plague  bacilli  grow  slowly.  They  usually  sink  to 
the  bottom  or  adhere  to  the  walls  of  the  tube  as  a  granular  deposit  and 
may  occasionally  form  a  deUcate  pelhcle.  Chain-formation  is  not  un- 
common. In  broth  cultures,  moreover,  a  peculiar  stalactite-like  growth 
is  often  seen,  when  the  culture  fluid  is  covered  with  a  layer  of  oil. 
Delicate  threads  of  growth  hang  down  from  the  surface  of  the  medium 
into  its  depths  like  stalactites.  Characteristic  involution  forms  are 
brought  out  best  when  the  bacilli  are  grown  upon  agar  containing  3 
per  cent  NaCl. 

Milk  is  not  coagulated.  In  litmus-milk  there  is  slight  acid  forma- 
tion. On  potato  and  on  blood  serum  the  growth  shows  nothing  char- 
acteristic or  of  differential  value.     On  pepton  media  no  indol  is  formed. 

Biological  Considerations. — Bacillus  pestis  is  aerobic.  Absence  of 
free  oxygen  is  said  to  prevent  its  growth,  at  least  under  certain  condi- 
tions of  artificial  cultivation.  It  is  non-motile.  Outside  of  the  animal 
body  the  bacilli  may  retain  viability  for  months  and  even  years  if 
preserved  in  the  dark  and  in  a  moist  environment.  In  cadavers  they 
may  live  for  weeks  and  months  if  protected  from  dryness.  In  pus  or 
sputum  from  patients  they  may  live  eight  to  fourteen  days.  These 
facts  are  of  great  hygienic  importance. 

Complete  drying  in  the  air  kills  the  bacilli  within  two  or  three  days.^ 
Thoroughly  dried  by  artificial  means  they  die  within  four  or  five  hours. 

Dry  heat  at  100°  C.  kills  the  bacillus  in  one  hour.^  Live  steam  or  boil- 
ing water  is  effectual  in  a  few  minutes.  The  bacilli  possess  great  resist- 
ance against  cold,  surviving  a  temperature  of  0°  C.  for  as  many  as 
forty  days. 

Direct  simlight  destroys  them  within  four  or  five  hours.  The  common 
disinfectants  are  effectual  in  the  following  strengths:  carboUc  acid,  one 
per  cent  kills  them  in  two  hours,  five  per  cent  in  ten  minutes;  bichloride 
of  mercury  1  : 1,000  is  effectual  in  ten  minutes. 

In  a  recent  communication  to  the  New  York  Pathological  Society, 
Dr.  Wilson  reported  that  plague  cultures  which  he  had  kept  sealed  for  as 
long  as  ten  years  in  the  ice  chest  were  found  living  and  virulent  at  the 
end  of  this  time.  This  ability  to  go  into  a  quasi  latent  stage  under 
suitable  conditions  is  of  the  greatest  importance  in  connection  with 
the  problem  of  prevention. 

1  Kitasato,  Lancet,  1894.  2  ^5^^^  Cgnt,  f^  Bakt.,  xxi,  1897. 


558  PATHOGENIC  MICROORGANISMS 

Pathogenicity. — In  man,  plague  is  acquired^  by  entrance  of  the  bacil- 
lus either  through  the  skin  or  by  the  respiratory  tract.  The  period  of 
incubation  is  about  three  to  seven  days.  Two  distinct  clinical  types 
of  the  disease  occur,  depending  upon  the  mode  of  infection.  When 
cutaneous  infection  has  occurred  the  disease  is  first  locaUzed  in  the 
lymph  nodes  nearest  the  point  of  inoculation.  If  the  respiratory  tract 
has  been  the  portal  of  entrance  the  disease  primarily  takes  the  form  of  a 
pneumonia. 

Infection  may  take  place  through  the  most  minute  lesions,  hardly 
visible  to  the  naked  eye.  Even  the  unbroken  skin  may  admit  the 
microorganisms  if  these  are  rubbed  in  with  sufficient  energy.  From  the 
primary  lymphatic  swellings,  the  bacilli  enter  the  blood  and  may  pro- 
duce secondary  foci. 

The  pneumonic  form  of  plague  usually  begins  with  symptoms  not 
unlike  a  typical  pneumonia  and  is  usually  fatal  within  four  or  five  or 
even  fewer  days.  This  form  of  the  disease  is  especially  menacing  as  a 
means  of  dissemination,  because  of  the  enormous  numbers  of  plague 
bacilU  in  the  sputum. 

One  of  the  chief  characteristics  of  the  general  systemic  plague  infec- 
tion is  the  very  marked  cardiac  depression. 

The  bacteriological  diagnosis  during  life  may  be  made  by  finding  the 
bacilli  in  the  sputum  or  in  aspiration  fluid  from  a  bubo.  The  micro- 
organisms are  identified  morphologically,  culturally,  by  animal  experi- 
ment, and  by  agglutination  reaction.  Blood  cultures  from  plague  ph- 
tients  often  yield  positive  results,  especially  when  the  blood  is  well 
diluted  in  neutral  broth  to  prevent  any  inhibiting  action  of  the  anti- 
bodies in  the  serum. 

At  autopsy,  in  man,  the  bacilli  are  found  in  the  primary  lesions,  in 
the  blood,  and  in  the  spleen,  the  liver,  and  the  lymphatics.  There  may 
be  hemorrhages  into  the  serous  cavities.  When  pneumonia  exists,  it 
usually  takes  the  form  of  a  bronchopneiunonia  with  extensive  swelling 
of  the  bronchial  lymph  nodes. 

In  cases  in  which  the  disease  is  prolonged,  there  are  often  tubercle- 
like foci  in  the  spleen,  liver,  and  lungs.  Histologically  these  foci  show 
central  necrosis  surrounded  by  the  usual  inflammatory  cell  reactions. 
In  more  chronic  cases  connective-tissue  encapsulation  may  appear. 

Bacillus  pestis  is  extremely  pathogenic  for  rats,  mice,  guinea-pigs, 
rabbits,  and  monkeys.     The  most  susceptible  of  these  animals  are  rats 

1  Gottschlich,  Zeit.  f .  Hyg.,  xxxv,  1900. 


BACILLUS  PESTIS  559 

and  guinea-pigs,  in  whom  mere  rubbing  of  plague  bacilli  into  the  un- 
broken skin  will  often  produce  the  disease.  This  method  of  experimen- 
tal infection  of  guinea-pigs  is  of  great  service  in  isolating  the  plague 
bacillus  from  material  contaminated  with  other  microorganisms.  For 
the  same  purpose,  infection  of  rats  subcutaneously  at  the  root  of 
the  tail  may  be  employed.  Such  inoculation  in  rats  is  invariably 
fatal. 

The  studies  of  McCoy  ^  upon  guinea-pigs  and  white  rats  show  that 
individual  plague  cultures  may  vary  considerably  in  virulence.  The 
size  of  the  dose,  always  excepting  enormous  quantities  such  as  a  whole 
agar  culture,  seems  to  make  little  difference  in  the  speed  with  which 
the  animals  die.  There  may  be  considerable  variation  in  the  suscep- 
tibility of  individual  animals.  Prolonged  cultivation  on  artificial  media 
may  gradually  reduce  the  virulence  of  plague  bacilli,  though,  as  stated 
above,  this  has  not  been  the  experience  of  all  observers. 

In  rats,  spontaneous  infection  with  plague  is  common  and  plays  an 
important  role  in  the  spread  of  the  disease.  Rats  become  infected  from 
the  cadavers  of  plague  victims  or  by  gnawing  the  dead  bodies  of  other 
rats  dead  of  the  disease.  The  pneumonic  type  of  the  disease  is  common 
in  these  animals  and  has  been  produced  in  them  by  inhalation  experi- 
ments. During  every  well-observed  plague  epidemic,  marked  mortality 
among  the  domestic  rats  has  been  noticed. 

Since  the  examination  of  rats  for  plague  is  an  important  phase  of 
the  study  of  epidemics,  it  may  be  well  to  review  the  typical  lesions  in 
these  animals  as  described  by  an  experienced  American  student  of  plague, 
George  W.  McCoy.^  McCoy,  agreeing  with  the  Indian  Plague  Com- 
mission, states  that  the  naked  eye  is  superior  to  the  microscopical  ex- 
amination. There  is  engorgement  of  the  subcutaneous  vessels  and  a 
pink  coloration  of  the  muscles.  The  bubo  when  present  is  sufficient 
for  diagnosis.  Marked  injection  surrounds  it  and  sometimes  there  is 
hemorrhagic  infiltration.  The  gland  itself  is  firm  but  usually  caseous  or 
occasionally  hemorrhagic.  In  the  liver  there  is  apparent  fatty  change, 
but  this  is  due  to  necrosis.  Pin-point  spots  give  it  a  stippled  appear- 
ance as  though  it  had  been  dusted  with  pepper.  Pleural  effusion  is  an 
important  sign.  The  spleen  is  large,  friable,  and  often  presents  pin- 
point granules  on  the  surface.  One  or  two  per  cent  of  rats  may  present 
no  gross  lesions.  Cultures  should  of  course  be  made.  The  method 
of  examination  consists  in  immersing  the  rat  in  any  convenient  antiseptic 


37 


1  McCoy,  Jour,  of  Inf.  Dis.,  vi,  1909. 

2  Gewge  W.  McCoy,  PubUc  Health  Reports,  July^  1912, 


560  PATHOGENIC  MICROORGANISMS 

to  kill  fleas  and  other  ectoparasites.  The  rats  are  nailed  by  their  feet 
to  a  shingle  and  the  skin  is  reflected  from  the  whole  front  of  the  body  and 
neck  so  as  to  expose  the  cervical,  axillary,  and  inguinal  regions.  The 
thoracic  and  abdominal  cavities  are  then  opened  and  examined. 

Wherry,^  McCoy ,2  and  others  have  found  that  the  California  ground 
squirrel  was  infected  with  plague,  during  the  recent  occurrence  of 
plague  on  the  Pacific  coast,  and  several  cases  of  plague  in  man  were 
traced  to  this  source.  In  studying  these  and  other  American  ro- 
dents McCoy  found  that  ground  squirrels  as  a  species  were  highly 
susceptible,  never  showing  natural  immunity.  Field  mice  were  but 
moderately  susceptible.  Gophers  were  highly  resistant.  McCoy  has 
also  described  a  case  of  spontaneous  infection  in  a  brush  rat  (Neo- 
toma  fuscipes).  Rock  squirrels  were  found  by  McCoy  to  be  readily 
infected. 

Wu  Lien  Teh  (G.  L.  Tuck)  ^  has  recently  found  that  the  Manchurian 
tarbagan  or  marmot  (Arctomys  bobac),  an  animal  trapped  for  its  fur, 
occasionally  suffers  from  plague.  The  disease  is  never  extensive  and  the 
animal  of  much  less  importance  in  spreading  the  disease  than  is  the  rat. 

The  two  principal  species  of  rats  to  be  considered  in  this  connection 
are  Epimys  norvegicus  and  Epimys  rattus.  The  spread  from  rat  to 
rat,  according  to  the  Second  Indian  Commission,  is  entirely  by  means 
of  infected  fleas. 

The  ordinary  spread  of  the  disease  to  man,  according  to  this  same 
commission,  comes  from  Epimys  rattus,  which  lives  in  close  relation- 
ship with  man  and  is  conveyed  to  man  largely  by  the  rat  flea,  Xenop- 
sylla  cheopis.  This  flea  leaves  the  dead  rat  in  about  three  days, 
and  is  capable  of  living  for  three  or  four  weeks  on  man's  blood. 
The  plague  bacilli  need  about  three  days'  incubation  in  the  body  of 
the  flea. 

Summarizing  the  knowledge  at  present  available  about  the  spread 
of  the  plague,  it  seems  likely  that,  excepting  in  the  case  of  pneumonic 
plague,  the  ordinary  method  is  by  means  of  rat  fleas. 

It  is  a  curious  fact  observed  by  various  bacteriologists  that  plague 
bacilli  isolated  from  pneumonic  cases  are  particularly  apt  to  cause 
pneumonic  lesions,  having,  as  it  were,  acquired  a  selective  pathogenicity 
for  the  lung.    A  most  valuable  contribution  to  our  knowledge  of  pneu- 


1  Wherry,  Jour.  Inf.  Dis.,  v,  1908. 

2  McCoy,  Jour.  Inf.  Dis.,  vi,  1909;  vii,  1910. 
5  Wu  Lien  Teh,  JouT.  of  Hyg.,  xiii,  1913. 


6ACILLUS  PESTIS  561 

monic  plague  has  recently  been  made  by  Strong,  Teague,  and  Barber ' 
in  their  report  of  the  American  Red  Cross  Expedition  to  Manchuria 
dm-ing  the  plague  epidemic  of  1910-11.  Their  investigations  were  made 
with  remarkable  courage  and  skill  under  difficult  conditions. 

The  chief  points  of  interest  in  their  reports  may  be  sunmiarized  as 
follows:  Expired  air  of  plague  patients  rarely  contains  the  bacilli;  these 
are  thrown  out  in  coughing  or  hawking.  Transmission  is,  in  this  form, 
direct  from  patient  to  patient  and  not  indirect  through  animals.  The 
first  localization  (Strong,  Teague,  and  Crowell)  is  in  the  bronchi  from 
which  extension  takes  place.  Septicemia  soon  follows  the  pneumonic 
process.  Spreading  occurs  most  likely  in  the  wet  and  cold  of  winter, 
since  the  bacteria  are  rapidly  destroyed  by  drying. 

Toxin  Formation. — The  systemic  symptoms  of  plague  are  largely  due 
to  the  absorption  of  poisonous  products  of  the  bacteria.  Albrecht  and 
Ghon,2  Wernicke,^  and  others  were  unable  to  obtain  any  toxic  action 
with  broth-culture  filtrates  and  concluded  that  the  poisons  of  B.  pestis 
were  chiefly  endotoxins,  firmly  attached  to  the  bacterial  body.  Kossel 
and  Overbeck,^  however,  on  the  basis  of  a  careful  investigation,  came 
to  the  conclusion  that,  in  addition  to  the  endotoxin,  there  is  formed  in 
older  broth  cultures  a  definite  and  important  true,  soluble  toxin.    • 

Immunization. — A  single  attack  of  plague  usually  protects  human 
beings  from  reinfection.  A  second  attack  in  the  same  individual  is 
extremely  rare.  Immunization  in  animals  produces  specific  agglutinat- 
ing and  bacteriolytic  substances  which  are  of  great  importance  in  the 
bacteriological  diagnosis  of  the  bacillus.  The  agglutinating  action  of  the 
serum  of  patients  is  clinically  important  in  the  diagnosis  of  the  disease, 
even  in  dilutions  of  one  in  ten,  since  undiluted  normal  human  serum 
has  no  agglutinating  effect  upon  plague  bacilli. 

Active  immunization  of  animals  ^  is  accomplished  by  inoculation  of 
the  whole  dead  bacteria.  Haffkine  has  attempted  active  immunization 
in  human  beings  by  subcutaneous  treatment  with  sterilized  broth  cul- 
tures of  B.  pestis.  Gaffky  ^  and  his  collaborators  recommend,  for  similar 
purposes,  forty-eight-hour  agar  cultures  of  a  bacillus  of  standard  viru- 
lence, emulsified  in  bouillon  and  sterilized  at  65°  C. 

1  Strong,  Teague,  and  Barber,  Philippine  Jour,  of  Sc,  Sect.  B,  vii,  1912,  No.  3. 

*  Albrecht  und  Ghon,  loc.  cit. 

3  Wernicke,  Cent.  f.  Bakt.,  Ref.,  xxiv,  1898.  ; 

*  Kossel  und  Overbeck,  Arb.  a.  d.  Gesundh.,  xviii,  1901. 

^  Yerdn,  Calmette,  et  Roux,  Ann.  de  I'inst.  Pasteur,  1895. 
^  Gaffky,  PfeiffeTy  Stickery  und  Dieudonne,  Aih.  a.  d.  kais.  Gesundheitsamt,  xvi, 
1899. 


562  PATHOGENIC   MICROORGANISMS 

The  curative  plague  serum  prepared  by  Yersin  and  others  by  the 
immunization  of  horses  with  plague  cultures  has  been  extensively  used 
in  practice  and  though  often  disappointing,  a  definitely  beneficial  in- 
fluence on  the  milder  cases  has  been  noted.  The  sera  are  standardized 
by  their  protective  power  as  measured  in  white  rats. 

THE  PLAGUE-LIKE  DISEASE  OF  RODENTS  (McCOY)  ^ 

Bacterium  Tularense  (McCoy  and  Chapin)^ 

McCoy  has  described  a  disease  occurring  in  Californian  ground 
squirrels  (Citellus  beechyi)  which  presents  lesions  very  similar  to  those 
of  plague  in  these  animals.  In  fact  the  disease  was  noticed  in  the 
course  of  the  systematic  examination  of  rodents  by  McCoy  at  the 
Federal  Laboratory  in  San  Francisco.  Although  McCoy  was  able  to 
transmit  the  disease  to  guinea-pigs,  mice,  rabbits,  monkeys,  and  gophers, 
and  plague-like  lesions  could  be  produced  in  most  of  the  animals,  he  was 
at  first  entirely  unable  to  cultivate  any  organism  from  these  lesions. 
In  1912  McCoy  and  Chapin  finally  succeeded  in  growing  the  specific 
bacterium  on  an  egg  medium  made  entirely  of  the  yolk.  Mor- 
phologically it  is  a  very  small  rod,  0.3  to  0.7  micron  in  length  and  often 
capsulated.  The  rods  stain  poorly  with  methylene  blue,  better  with 
carbol  fuchsin  or  gentian  violet.  They  are  found  in  large  numbers  in 
the  spleens  of  animals  dead  of  the  disease. 

1  McCoy,  U.  S.  Public  Health  Bull.  43,  1911. 

2  McCoy  and  Chapin,  Jour,  of  Inf.  Dis.,  x,  1912. 


CHAPTER  XL 
BACILLUS  ANTHRACIS  AND  ANTHRAX 
{Milzbrand,  Charhon)     . 

Anthrax  is  primarily  a  disease  of  the  herbivora,  attacking  especially 
cattle  and  sheep.  Infection  not  infrequently  occurs  in  horses,  hogs,  and 
goats.  In  other  domestic  animals  it  is  exceptional.  Man  is  susceptible  to 
the  disease  and  contracts  it  either  directly  from  the  living  animals  or 
from  the  hides,  wool,  or  other  parts  of  the  cadaver  used  in  the  industries. 

The  history  of  the  disease  dates  back  to  the  most  ancient  periods  and 
anthrax  has,  at  all  times,  been  a  severe  scourge  upon  cattle-  and  sheep- 
raising  communities.  Of  all  infections  attacking  the  domestic  animals 
no  other  has  claimed  so  many  victims  as  anthrax.  In  Russia,  where 
the  disease  is  most  common,  72,000  horses  are  .said  to  have  succumbed 
in  one  year  (1864)  .^ 

In  Austro-Hungary,  Germany,  France,  and  the  Eastern  countries, 
each  year  thousands  of  animals  and  numerous  human  beings  perish  of 
anthrax.  In  England  and  America  the  disease  is  relatively  infrequent. 
No  quarter  of  the  globe,  however,  is  entirely  free  from  it. 

Especial  historical  interest  attaches  to  the  anthrax  bacillus  in  that 
it  was  the  first  microorganism  proved,  definitely  to  bear  a  specific  etio-* 
logical  relationship  to  an  infectious  disease.  The  discovery  of  the  an- 
thrax bacillus,  therefore,  laid,  as  it  were,  the  cornerstone  of  modem 
bacteriology.  The  bacillus  was  first  observed  in  the  blood  of  infected 
animals  by  PoUender  in  1849,  and,  independently,  by  Brauell  in  1857. 
Davaine,^  however,  in  I'SBS,  was  the  first  one  to  produce  experimental 
infection  in  animals  with  blood  containing  the  bacilli  and  to  suggest 
a  direct  etiological  relationship  between  the  two.  Final  and  absolute 
proof  of  the  justice  of  Davaine's  contentions,  however,  was  not  brought 
until  the  further  development  of  bacteriological  technique,  by  Koch,^ 
had  made  it  possible  for  this  last  observer  to  isolate  the  bacillus  upon 

1  Quoted  from  Sobernheim,  Kplle  und  Wassermann.,  vol.  ii. 

2  Davaine,  Comptes  rend,  de  I'acad.  des  sci.,  Ivii,  1863. 
»  Koch,  Cohn's  ''  Beitr.  z.  Biol.  d.  Pflanzen,"  ii,  1876. 

563 


564 


PATHOGENIC  MICROORGANISMS 


artificial  media  and  to  reproduce  the  disease  experimentally  by  inocu- 
lation with  pure  cultures. 

Morphology  and  Staining. — The  anthrax  bacillus  i^  a  straight  rod, 
5  to  10  micra  in  length,  1  to  3  micra  in  width.  It  is  non-motile. 
In  preparations  made  from  the  blood  of  an  infected  animal,  the  bacilli 
are  usually  single  or  in  pairs.  Grown  on  artificial  media  they  form 
tangles  of  long  threads.    Their  ends  are  cut  off  squarely,  in  sharp  con- 


FiG.  120. — Bacillus  anthracis.    From  pure  culture  on  agar. 


trast  to  the  rounded  ends  of  many  other  bacilli.  The  comers  are  often 
sharp  and  the  ends  of  bacilli  in  contact  in  a  chain  often  touch  each  other 
only  at  these  points,  leaving  in  consequence  an  oval  chink  between  the 
ends  of  the  organisms.  The  appearance  of  a  chain  of  anthrax  bacilli, 
therefore,  has  been  not  inaptly  compared  to  a  rod  of  bamboo.  On 
artificial  media  the  anthrax  bacillus  forms  spores.  Oxygen  is  necessary 
for  the  formation  of  these  spores  and  they  are  consequently  not  found 


BACILLUS  ANTHRACIS  AND  ANTHRAX  565 

in  the  blood  of  infected  subjects.  The  spores  are  located  in  the  middle 
of  the  bacilli  and  are  distinctly  oval.  They  are  difficult  to  stain,  but 
may  be.  demonstrated  by  any  of  the  usual  spore-staining  procedures, 
such  as  Moller's  or  Novy's  methods.  The  bacilH  themselves  are  easily 
stained  by  the  usual  anilin  dyes,  and  gentian-violet  or  fuchsm  in  aque- 
ous solution  may  be  conveniently  employed.  They  are  not  decolorized 
by  Gram's  method. 

In  preparations  from  animal  tissues  or  blood,  stained  by  special  pro- 


FiG.     121. — Bacillus    anthkacis.      In    section    of    kidney  of  animal    dead    of 

anthrax. 

cedures,  the  anthrax  bacillus  may  occasionally  be  seen  to .  possess  a  cap- 
sule. The  capsule  is  never  seen  in  preparations  from  the  ordinary 
artificial  media.  Some  observers  have  demonstrated  them  in  cultures 
grown  in  fluid  blood  serum.  In  chains  of  anthrax  bacilli,  the  capsule 
when  present  seems  to  envelop  the  entire  chain  and  not  the  individual 
bacteria  separately. 

Isolation. — Isolation  of  the  anthrax  bacillus  from  infected  material 


566 


PATHOGENIC  MICROORGANISMS 


is  comparatively  simple,  both  because  of  the  ease  of  its  cultivation  and 
because  of  the  sharply  characteristic  features  of  its  morphological  and 
cultural  appearance. 

Cultivation. — The  anthrax  bacillus  is  an  aerobic,  facultatively  anaero- 
bic bacillus.  While  it  may  develop  slowly  and  sparsely  under  anaerobic 
conditions,  free  oxygen  is  required  to  permit  its  luxuriant  and  charac- 
teristic growth. 

The  optimum  temperature  for  its  cultivation  ranges  about  37.5°  C. 
It  is  not,  however,  delicately  susceptible  to  moderate  variations  of  tem- 


Fig.  122. — Bacillus  anthracis.     In  smear  of  spleen  of  animal  dead  of  anthrax. 


perature  and  growth  does  not  cease  until  temperatures  as  low  as  12°  C. 
or  as  high  as  45°  C.  are  reached.  By  continuous  cultivation  at  some  of 
the  temperatures  near  either  the  higher  or  the  lower  of  these  limits,  the 
bacillus  may  become  well  adapted  to  the  new  environment  and  attain 
luxuriant  growth.^ 

The  anthrax  bacillus  may  be  cultivated  on  all  of  the  usual  artificial 
media,  growing  upon  the  meat-extract  as  well  as  upon  the  meat-infusion 
media. 


1  Dieudonne,  Arb.  a.  d.  kais.  Gesundheitsamt,  1894. 


BACILLUS    ANTHRACIS    AND    ANTHRAX  567 

It  may  be  cultivated  also  upon  hay  infusion,  various  other  vegetable 
media,  sugar  solutions,  and  urine.  While  moderate  acidity  of  the 
medium  does  not  prevent  the  growth  of  this  bacillus,  the  most  favorable 
reaction  for  media  is  neutrality  or  slight  alkalinity. 

On  gelatin  plates,  colonies  develop  within  twenty-four  to  forty-eight 
hours  as  opaque,  white  disks,  pin-head  in  size,  irregularly  round  and 
rather  flat.  As  the  colonies  increase  in  size  their  outlines  become  less 
regular  and  under  the  microscope  they  are  seen  to  be  made  up  of  a 
hair-like  tangle  of  threads  spreading  in  thin  wavy  layers  from  a  more 
compact  central  knot.  The  microscopic  appearance  of  these  colonies 
has  been  aptly  described  as  resembling  a  Medusa  head.    Fragments  of  a 


rv^>^'5^5 


Fig.  123. — ^Anthrax  Colony  on  Gelatin.      (After  Giinther.) 

colony  examined  on  a  slide  with  a  higher  power  show  the  individual 
threads  to  be  made  up  of  parallel  chains  of  bacilli. 

After  a  day  or  two  of  further  growth,  the  gelatin  about  the  colonies 
becomes  fluidified. 

In  gelatin  stab  cultures,  growth  appears  at  first  as  a  thin  white  line 
along  the  course  of  the  puncture.  From  this,-  growth  proceeds  in  thin 
spicules  or  filaments  diverging  from  the  stab,  more  abundantly  near  the 
top  than  near  the  bottom  of  the  stab,  owing  to  more  active  growth  in 
well  oxygenated  environment.  The  resulting  picture  is  that  of  a  small 
inverted  "Christmas  tree.''  Fluidification  begins  at  the  top,  at  first  a 
shallow  depression  filled  with  an  opaque  mixture  of  bacilli  and  fluid. 


568 


PATHOGENIC  MICROORGANISMS 


Later  the  bacilli  sink  to  the  bottom  of  the  flat  depression,  leaving  a  clea? 
supernatant  fluid  of  peptonized  gelatin. 

In  hroth,  growth  takes  place  rapidly,  but  does  not  lead  to  an  even, 
general  clouding.  There  is  usually  an  initial  pellicle  formation  at  the 
top  where  the  oxygen  supply  is  greatest.  Simultaneously  with  this  a 
slimy  mass  appears  at  the  bottom  of  the  tube,  owing  to  the  sinking  of 


Fig.  124. — Anthrax  Colony  on  Agar. 


bacilli  to  the  bottom.  Apart  from  isolated  flakes  and  threads  the  inter- 
vening broth  is  clear.  Shaken  up,  the  tube  shows  a  tough,  stringy  mass, 
not  unlike  a  small  cotton  fluff,  and  general  clouding  is  produced  only 
by  vigorous  mixing. 

Upon  a^ar  plates,  growth  at  37.5°  C.  is  vigorous  and  colonies  appear 


BACILLUS  ANTHRACIS   AND  ANTHRAX  569 

within  twelve  to  twenty-four  hours.  They  are  irregular  in  outline, 
slightly  wrinkled,  and  show  under  the  microscope  the  characteristic 
tangled-thread  appearance  seen  on  gelatin,  except  that  they  are  more 
compact  than  upon  the  former  medium.  The  colonies  are  slightly  glisten- 
ing and  tough  in  consistency. 

On  agar  slants,  the  colonies  usually  become  confluent,  the  entire 
surface  soon  being  covered  by  a  grayish,  tough  pellicle  which,  if  fished, 
has  a  tendency  to  come  away  in  thin  strips  or  strands. 

On  potato,  growth  is  rapid,  white,  and  rather  dry.  Sporulation  upon 
potato  is  rapid  and  marked,  and  the  medium  is  favorable  for  the  study 
of  this  phase  of  development. 

Milk  is  slowly  acidified  and  slowly  coagulated.  This  action  is  chiefly 
upon  the  casein ;  very  few,  if  any,  changes  being  produced  either  in  the 
sugars  or  in  the  fats  of  the  milk.  The  acids  formed  are,  according  to 
Iwanow,^  chiefly  formic,  acetic,  and  caproic  acids. 

Biological  Considerations. — The  anthrax  bacillus  is  aerobic  and  facul- 
tatively anaerobic.  It  is  non-motile  and  possesses  no  flagella.  In  the 
animal  body  it  occasionally  forms  capsules.  In  artificial  cultures  in 
the  presence  of  oxygen,  it  sooner  or  later  invariably  forms  spores.  The 
spores  appear  after  the  culture  has  reached  its  maximum  of  develop- 
ment. Sporulation  never  occurs  in  the  animal  body,  probably  because 
of  the  absence  of  sufficient  free  oxygen.  Spores  are  formed  most  exten- 
^vely  ^  at  temperatures  ranging  from  20°  C.  to  30°  C.  Spore  formation 
ceases  below  18°  C.  and  above  42°  C.  For  different  strains  these  figures 
may  vary  slightly,  as  has  been  shown  from  the  results  of  variouS 
observers.  Spores  appear  most  rapidly  and  regularly  upon  agar  and 
potato  media. 

The  spore — one  in  each  bacillus — appears  as  a  small,  highly  refractile 
spot  in  the  center  of  the  individual  bacterium.  As  this  enlarges,  the 
body  of  the  bacillus  around  it  gradually  undergoes  granular  degenera- 
tion and  loses  its  staining  capacity.^ 

If  anthrax  bacilli  are  cultivated  for  prolonged  periods  upon  media 
containing  hydrochloric  or  rosolic  acid  or  weak  solutions  of  carbolic 
acid,*  cultures  may  be  obtained  which  do  not  sporulate  and  which  seem 
permanently  to  have  lost  this  power,  without  losing  their  virulence  to 
the  same  degree.     Similar  results  may  be  obtained  by  continuous  cul- 

1  Iwanow,  Ann.  de  I'inst.  Pasteur,  1892. 

2  Koch,  loc.  cit. 

3  Behring,  Zeit.  f.  Hyg.,  vi  and  vii,  1889;  Deut.  med.  Woch.,  1889. 

*  Chamberland  et  Roux,  Comptes  rend,  de  I'acad.  des  sci.,  xcvi,  1882. 


570  PATHOGENIC  MICROORGANISMS 

tivation  at  temperatures  above  42°  C.  By  this  procedure,  however, 
virulence,  too,  is  considerably  diminished. 

Resistance. — Because  of  its  property  of  spore  formation,  the  anthrax 
bacillus  is  extremely  resistant  toward  Chemical  and  physical  environ- 
ment. The  vegetative  forms  themselves  are  not  more  resistant  than 
most  other  npn-sporulating  bacteria,  being  destroyed  by  a  temperature 
of  54°  C.  in  ten  minutes.  Anthrax  spores  may  be  kept  in  a  dry  state 
for  many  years  without  losing  their  viability.^  While  different  strains 
of  .anthrax  spores  show  some  variation  in  their  powers  of  resistance, 
all  races  show  an  extremely  high  resistance  to  heat.  Dry  heat  at  140° 
C.  kills  them  only  after  three  hours.^  Live  steam  at  100°  kills  them  in 
five  to  ten  minutes.  Boiling  in  water  destroys  them  in  about  ten  min- 
utes. Low  temperatures  have  but  little  effect  upon  them.  Ravenel  ^ 
found  that,  frozen  by  liquid  air,  they  were  still  viable  after  three  hours. 

The  variability  shown  by  different  strains  of  spores  in  their  resistance 
to  heat  is  even  more  marked  in  their  behavior  toward  chemicals.^  Some 
strains  will  retain  their  viability  after  exposure  to  five-per-cent  carbolic 
acid  for  forty  days,^  while  others  are  destroyed  by  the  same  solution  in 
two  days.  Corrosive  sublimate,  1  :  2,000,  kills  most  strains  of  anthrax 
in  forty  minutes. 

Direct  sunlight  destroys  anthrax  spores  within  six  to  twelve  hours.^ 

Pathogenicity. — ^The  anthrax  bacillus  is  pathogenic  for  cattle, 
sheep,  guinea-pigs,  rabbits,  rats,  and  mice.  The  degrees  of  susceptibil- 
ity of  these  animals  differ  greatly,  variations  in  this  respect  existing  even 
among  different  members  of  the  same  species.  Thus,  the  long-haired 
Algerian  sheep  show  a  high  resistance,  while  the  European  variety  are 
highly  susceptible;  and,  similarly,  the  gray  rat  is  much  more  resistant 
than  the  white  rat.  Dogs,  hogs,  cats,  birds,  and  the  cold-blooded  ani- 
mals are  relatively  insusceptible.  For  man  the  bacillus  is  definitely 
pathogenic,  though  less,  so  than  for  some  of  the  animals  mentioned 
above. 

While  separate  races  of  anthrax  bacilli  may  vary  much  in  their  de- 
gree of  virulence,  a  single  individual  strain  remains  fairly  constant  in 
this  respect  if  preserved,  dried  upon  threads  or  kept  in  sealed  tubes,  in 

1  Surmont  et  Arnould,  Ann.  de  Tinst.  Pasteur,  1894. 

2  Koch  und  Wolff hiigel,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1881. 
»  Ravenel,  Medical  News,  vii,  1899. 

*  Frankel,  Zeit.  f.  Hyg.,  vi,  1889. 
'  Koch,  loc.  cit. 

•  Momont,  Ann.  de  Finst.  Pasteur,  1892. 


BACILLUS  ANTHRACIS  AND  ANTHRAX  571 

a  cold,  dark  place.  Virulence  may  be  reduced  ^  by  various  attenuating 
laboratory  procedures  which  are  of  importance  in  that  they  have  made 
possible  prophylactic  immunization.  Heating  the  bacilli  to  55°  C.  for 
ten  minutes  considerably  reduces  their  virulence.  Similar  results  are 
obtained  by  prolonged  cultivation  at  temperatures  of  42°  to  43°  C, 
or  by  the  addition  of  weak  disinfectants  to  the  culture  fluids.^  Once 
reduced,  the  new  grade  of  virulence  remains  fairly  constant.  Increase 
of  virulence  may  be  artificially  produced  by  passage  through  animals. 

Experimental  infections  in  susceptible  animals  are  most  easily  accom- 
plished by  subcutaneous  inoculations.  The  inoculation  is  followed,  at 
first,  by  no  morbid  symptoms,  and  some  animals  may  appear  perfectly 
well  and  comfortable  until  within  a  few  hours  or  even  moments  before 
death,  when  they  suddenly  become  visibly  very  ill,  rapidly  go  into 
collapse,  and  die.  The  length  of  the  disease  depends  to  some  extent, 
of  course,  upon  the  resistance  of  the  infected  subject,  being  in  guinea- 
pigs  and  mice  from  twenty-four  to  forty-eight  hours.  The  quantity  of 
infectious  material  introduced,  on  the  other  hand,  has  little  bearing 
upon  the  final  outcome,  a  few  bacilli,  or  even  a  single  bacillus,  often 
sufficing  to  bring  about  a  fatal  infection.  Although  the  bacilli  are  not 
demonstrable  in  the  blood  until  just  before  death,  they  nevertheless 
invade  the  blood  and  lynfph  streams  immediately  after  inoculation, 
and  are  conveyed  by  these  to  all  the  organs.  This  has  been  demonstrated 
clearly  by  experiments  where  inoculations  into  the  tail  or  ear  were  im- 
mediately followed  by  amputation  of  the  inoculated  parts  without  pre- 
vention of  the  fatal  general  infection.  The  bacilli  are  probably  not  at 
first  able  to  multiply  in  the  blood.  At  the  place  of  inoculation  and 
probably  in  the  organs  they  proliferate,  until  the  resistance  of  the  in-, 
fected  subject  is  entirely  overcome.  At  this  stage  of  the  disease,  no 
longer  held  at  bay  by  any  antagonistic  qualities  of  the  blood,  they  enter 
the  circulation  and  multiply  within  it.  Autopsy  upon  such  animals 
reveals  an  edematous  hemorrhagic  infiltration  at  the  point  of  inocu- 
lation. The  spleen  is  enlarged  and  congested.  The  kidneys  are  con- 
gested, and  there  may  be  hemorrhagic  spots  upon  the  serous  mem- 
branes. The  bacilli  are  found  in  large  numbers  in  the  blood  and  in  the 
capillaries  of  all  the  organs. 

The  mode  of  action  of  Bacillus  anthracis  is  as  yet  an  unsettled  point. 
It  is  probable  that  death  is  brought  about  to  a  large  extent  by  purely 

'  Toussaint,  Comptes  rend,  de  Tacad.  des  sci.,  xci,  1880;  Pasteur,  Chamberland 
et  Roux,  Comptes  rend,  de  I'acad.  des  sci.,  xcii,  1881. 
2  Chamberland  et  Roux,  ibid.,  xevi,  188?, 


572  PATHOGENIC  MICROORGANISMS 

mechanical  means,  such  as  capillary  obstruction.  Neither  a  true 
secretory  toxin  nor  an  endotoxin  has  been  demonstrated  for  the  anthrax 
bacillus.  The  decidedly  toxemic  clinical  picture  of  the  disease,  however, 
in  some  animals  and  in  man,  precludes  our  definitely  concluding  that 
such  poisons  do  not  exist.  It  is  a  matter  of  fact,  however,  that  neither 
culture  filtrates  nor  dead  bacilli  have  any  noticeable  toxic  effect  upon 
test  animals,  and  exert  no  appreciable  immunizing  action. 

Spontaneous  infection  of  animals  takes  place  largely  by  way  of  the 
alimentary  canal,  the  bacilli  being  taken  in  with  the  food.  The  bacteria 
are  swallowed  as  spores,  and  therefore  resist  the  acid  gastric  juice.  In 
the  intestines  they  develop  into  the  vegetative  forms,  increase,  and 
gradually  invade  the  system.  The  large  majority  of  cattle  infections 
are  of  this  type.  Direct  subcutaneous  infection  may  also  occur  sponta- 
neously when  small  punctures  and  abrasions  about  the  mouth  are  made 
by  the  sharp  spicules  of  the  hay,  straw,  or  other  varieties  of  fodder. 

When  infection  upon  a  visible  part  occurs,  there  is  formed  a  diffuse, 
tense  local  swelling,  not  unlike  a  large  carbuncle.  The  center  of  this  may 
be  marked  by  a  black,  necrotic  slough,  or  may  contain  a  pustular  de- 
pression. 

Infection  by  inhalation  is  probably  rare  among  animals.  Trans- 
mission among  animals  is  usually  by  the  agency  of  the  excreta  or  un- 
bumed  carcasses  of  infected  animals.  The  bacilli  escaping  from  the 
body  are  deposited  upon  the  earth  together  with  animal  and  vegetable 
matter,  which  forms  a  suitable  medium  for  sporulation.  The  spores 
may  then  remain  in  the  immediate  vicinity,  or  may  be  scattered  by 
rain  and  wind  dver  considerable  areas.  The  danger  from  buried  car- 
casses, at  first  suspected  by  Pasteur,  is  probably  very  slight,  owing  to  the 
fact  that  the  bacilli  can  not  sporulate  in  the  anaerobic  environment  to 
which  the  burying-process  subjects  them.  The  disease,  in  infected  cattle 
and  sheep,  is  usually  acute,  killing  within  one  or  two  days.  The  mortality 
is  extremely  high,  fluctuating  about  eighty  per  cent. 

In  man  the  disease  is  usually  acquired  by  cutaneous  inoculation.  It 
may  also  occur  by  inhalation  and  through  the  alimentary  trabt. 

Cutaneous  inoculation  occurs  usually  through  small  abrasions  or 
scratches  upon  the  skin  in  men  who  habitually  handle  live-stock,  and 
in  butchers,  or  tanners  of  hides.  Infection  occurs  most  frequently  upon 
the  hands  and  forearms.  The  primary  lesion,  often  spoken  of  as  "  malig- 
nant pustule,"  appears  within  twelve  to  twenty-four  hours  after  inocula- 
tion, and  resembles,  at  first,  an  ordinary  small  furuncle.  Soon,  however, 
its  center  will  show  a  vesicle  filled  with  sero-sanguineous,  later  sero 


BACILLUS  ANTHRACIS  AND  ANTHRAX  573 

purulent  fluid.  This  may  change  into  a  black  central  necrosis  sur- 
rounded by  an  angry  red  edematous  areola.  Occasionally  local  gangrene 
and  general  systemic  infection  may  lead  to  death  within  five  or  six  days. 
More  frequently,  however,  especially  if  prompt  excision  is  practiced,  the 
patient  recovers.  The  early  diagnosis  of  the  condition  is  best  made 
bacteriologically  by  finding  the  bacilli  in  the  local  discharge. 

The  pulmonarj^  infection,  known  as  "wool-sorter's  disease,"  occurs 
in  persons  who  handle  raw  wool,  hides,  or  horse  hair,  by  the  inhala- 
tion or  by  the  swallowing  of  spores.  The  disease  is  fortunately  rare  in 
this  country.  The  spores,  once  inhaled,  develop  into  the  vegetative 
forms  ^  and  .  these  travel  along  the  lymphatics  into  the  lungs  and 
pleura.  The  disease  manifests  itself  as  a  violent,  irregular  pneumonia, 
which,  in  the  majority  of  cases,  leads  to  death.  The  bacilli  in  these 
cases  can  often  be  found  in  the  sputum  before  death. 

Infection  through  the  alimentary  canal  may  occasionally,  though 
rarely,  occur  in  man,  the  source  of  infection  being  usually  ingestion  of 
the  uncooked  meat  of  infected  animals.  This  form  of  infection  is  rare, 
because  in  many  cases  the  bacilli  have  not  sporulated  in  the  animal 
and  the  ingested  vegetative  forms  are  injured  or  destroyed  by  the  acid 
gastric  juice.  When  viable  spores  enter  the  gut,  however,  infection  may 
take  place,  the  initial  lesion  being  localized  usually  in  the  small  intes- 
tine. The  clinical  picture  that  follows  is  one  of  violent  enteritis  with 
bloody  stools  and  great  prostration.  Death  is  the  rule.  The  diagnosis 
is  made  by  the  discovery  of  the  bacilli  in  the  feces. 

General  hygienic  prophylaxis  against  anthrax  consists  chiefly  in  the 
destruction  of  infected  animals,  in  the  burying  of  cadavers,  and  in  the 
disinfection  of  stables,  etc.  The  practical  impossibility  of  destroying, 
the  anthrax  spores  in  infected  pastures,  etc.,  makes  it  necessary  to  re- 
sort to  prophylactic  immunization  of  cattle  and  sheep. 

Immunity  against  Anthrax. — Minute  quantities  of  virulent  anthrax 
cultures  usually  suffice  to  produce  death  in  susceptible  animals.  Dead 
cultures  are  inefficient  in  calling  forth  any  immunity  in  treated  subjects. 
It  is  necessary,  therefore,  for  the  production  of  active  immunity  to 
resort  to  attenuated  cultures.  The  safest  way  to  accomplish  such  at- 
tenuation is  the  one  originated  by  Pasteur,^  consisting  in  prolonged 
cultivation  of  the  bacillus  at  42°  to  43°  C.  in  broth.  Non-spore-forming 
races  are  thus  evolved. 

The  longer  the  bacilli  are  grown  at  the  above  temperature  the  greater 

I  Epjringer,  Wieri.  med.  Woch.,  1888.        2  Pasteur,  loc.  dt. 


574  PATHOGENIC  MICROORGANISMS 

is  the  reduction  in  their  virulence.  Koch,  Gaffky,  and  Loeffler/  utilizing 
the  variations  in  susceptibilities  of  different  species  of  animals,  devised  a 
method  by  means  of  which  the  relative  attenuation  of  a  given  culture  may 
be  estimated  and  standardized.  Rabbits  are  less  susceptible  than  guinea- 
pigs,  and  virulent  anthrax  cultures,  grown  for  two  or  three  days  under  the 
stated  conditions,  lose  their  power  to  kill  rabbits,  but  are  less  virulent  for 
guinea-pigs.  After  ten  to  twenty  days  of  further  cultivation  at  42°  C. 
the  virulence  for  the  guinea-pig  disappears,  but  the  culture  is  potent 
against  the  still  more  susceptible  mouse.  Even  the  virulence  for  mice 
may  be  entirely  eliminated  by  further  cultivation  at  this  temperature. 

The  method  of  active  immunization  first  practiced  by  Pasteur,  and 
still  used  extensively,  is  carried  out  as  follows:  Two  ianthrax  cultures 
of  varjnng  degrees  of  attenuation  are  used  as  vaccins.  The  premier 
vaccin  is  a  culture  which  has  lost  its  virulence  for  guinea-pigs  and 
rabbits,  and  is  potent  only  against  mice.  The  deuxieme  vaccin  is  a  cul- 
ture which  is  still  definitely  virulent  for  mice  and  guinea-pigs,  but  not 
potent  for  rabbits.  Forty-eight-hour  broth  cultures  of  these  strains, 
grown  at  37.5°  C,  form  the  vaccin  actually  employed.  Vaccin  I  is 
subcutaneously  injected  into  cattle  in  doses  of  0.25  c.c,  sheep  receiving 
about  half  this  quantity.  After  twelve  days  have  elapsed  similar  quan- 
tities of  Vaccin  II  are  injected. 

Pasteur's  method  has  given  excellent  results  and  confei-s  an  im- 
munity which  lasts  about  a  year. 

Chauveau  ^  has  modified  Pasteur's  method  by  growing  the  bacilli 
in  bouillon  at  38°  to  39°  C,  at  a  pressure  of  eight  atmospheres.  Cul- 
tures are  then  made  of  races  attenuated  in  this  way,  upon  chicken 
bouillon  and  allow^ed  to  develop  for  thirty  days.  Single  injections  of 
0.1  c.c.  each  of  such  cultures  are  said  to  protect  cattle. 

Active  immunization  of  small  laboratory  animals  is  very  difficult, 
but  can  be  accomplished  by  careful  treatment  with  extremely  attenu- 
ated cultures. 

Passive  immunization  by  means  of  the  serum  of  actively  immune 
animals  was  first  successfully  accomplished  by  Sclavo.^ 

The  subject  of  passive  immunization  has  been  especially  investigated 
and  practically  applied  by  Sobernheim.^  The  serum  used  is  produced  by 
actively  immunizing  sheep.    It  is  necessary  to  carry  immunization  to  an 

1  Koch,  Gaffky,  und  Loeffler,  Mitt.  a.  d.  kais.  Gesundheitsamt,  1884. 

2  Chauveau,  Comptes  rend,  de  I'acado  des  sci.,  1884. 

3  Sclavo,  Cent.  f.  Bakt.,  xviii,  1895. 

«  Sobemheim,  Zeit.  f.  Hyg.,  xxv,  1897;  xxxi,  1899. 


BACILLUS   ANTHRACIS    AND   ANTHRAX  575 

extremely  high  degree  in  order  to  obtain  any  appreciable  protective 
power  in  the  serum.  This  is  accomplished  by  preliminary  treatment 
with  Pasteur's  or  other  attenuated  vaccines,  followed  by  gradually 
increasing  doses  of  fully  virulent  cultures.  Treatment  continued  at 
intervals  of  two  weeks,  for  two  or  three  months,  usually  produces  an 
effective  serum.  Horses  and  cattle  may  also  be  used  for  the  process,  but 
they  are  believed  by  Sobernheim  to  give  less  active  sera  than  sheep. 
Bleeding  is  done  about  three  weeks  after  the  last  injection.  The  sera 
are  stable  and  easily  preserved. 

Injections  of  20  to  25  c.c.  of  such  a  serum  have  been  found  to  protect 
animals  effectually  from  anthrax  and  to  confer  an  immunity  lasting 
often  as  long  as  two  months.  Animals  already  infected  are  said  to  be 
saved  by  treatment  with  25  to  100  c.c.  of  the  serum. 

Neither  specific  bactericidal  nor  bacteriolytic  properties  have,  so 
far,  been  demonstrated  in  these  immune  sera.  In  fact,  these  properties 
are  distinctly  more  pronounced  against  Bacillus  anthracis  in  the  normal 
sera  of  rats  and  dogs.  Agglutinins  have  not  been  satisfactorily  demon- 
strated in  sera,  partly  because  of  the  great  technical  difficulties  en- 
countered in  the  active  chain-formation  of  the  bacillus  in  fluid  media. 
An  increase  of  opsonic  power  of  such  serum  over  normal  serum  has 
not  been  satisfactorily  demonstrated. 

Bacteria  Closely  Resembling  Bacillus  anthracis. — In  most  laboratory 
collections  there  are  strains  of  true  anthrax  bacilli  so  attenuated  that 
they  are  practically  non-pathogenic.  These  do  not  differ  from  the 
virulent  strains  in  any  morphological  or  cultural  characteristics. 
Besides  such  strains  there  are  numerous  non-virulent  bacteria  culturally 
not  identical  with  Bacillus  anthracis,  but  resembling  it  very  closely. 

B.  ANTHRACOiDEs  {Hueppe  and  Wood  ^) . — A  Gram-positive  bacillus, 
morphologically  different  from  B.  anthracis  in  that  the  ends  are  more 
rounded.  Culturally,  somewhat  more  rapid  in  growth  and  more  rapid 
in  gelatin  fluidification.  Non-pathogenic.  Otherwise  indistinguishable 
from  B.  anthracis. 

B.  RADicosus  (Wurzel  Bacillus) . — Cultivated  from  water — city  water 
supplies.  Morphologically  somewhat  larger  than  Bacillus  anthracis,  and 
the  individual  bacilli  more  irregular  in  size.  Very  rapid  fluidification  pf 
gelatin  and  growth  most  active  at  room  temperature.     Non-pathogenic. 

B.  suBTiLis  {Hay  Bacillus). — Although  not  very  closely  related  tc 
the  anthrax  group,  this  bacillus  is  somewhat  similar  and  conveniently 

» Hueppe  und  Wood,  Berl.  klin.  Woch.,  xvi,  1889. 


576 


PATHOGENIC  MICROORGANISMS 


described  in  this  connection.  It  is  of  importance  to  workers  with  patho- 
genic bacteria,  because  of  the  frequency  with  which  it  is  found  as  a 
saprophyte  or  secondary  invader  in  chronic  suppurative  lesions. 

Morphology. — Straight  rod,  2  to  8  micra  long,  0,7  micron  wide.  Spores 
formed  usually  slightly  nearer  one  pole  than  the  other.  Grows  in  long 
chains  and  only  in  such  chains  are  spores  found.  It  does  not  decolor- 
ize by  Gram's  method.     Is  actively  motile  in  young  cultures  in  which 


Fig.  125. — Bacillus  Subtilis.     (Hay  Bacillus.) 


the  bacilli  are  single  or  in  pairs.  In  older  cultures  chains  are  formed 
and  the  bacilli  become  motionless.  Gelatin  is  liquefied.  On  gelatin 
and  agar  the  bacilli  grow  as  a  dry  corrugated  pellicle.  Microscopically, 
the  colonies  are  made  up  of  interlacing  threads,  being  irregularly  round 
with  fringed  edges.  There  is  a  tendency  to  confluence.  The  bacillus 
is  found  in  brackish  water,  infusions  of  vegetable  matter,  etc.,  and  is 
practically  non-pathogenic,  occurring  only  occasionally  as  a  saprophyte 
in  old  sinuses  and  infected  wounds. 


CHAPTER  XLI 
BACILLUS  PYOCYANEUS 

It  is  a  matter  of  common  surgical  experience  that  many  suppurating 
wounds,  especially  sinuses  of  long  standing,  discharge  pus  which  is  of  a 
bright  green  color.  The  fact  that  this  peculiar  type  of  purulent  inflam- 
mation is  due  to  a  specific  chromogenic  microorganism  was  first  demon- 
strated by  Gessard  ^  in  1882.  The  bacillus  which  was  described  by  Ges- 
sard  has  since  become  the  subject  of  much  careful  research  and  has  been 
shown  to  hold  a  not  unimportant  place  among  pathogenic  bacteria.^ 

Morphology  and  Staining. — Bacillus  pyocyanieus  is  a  short  rod,  usu- 
ally straight,  occasionally  slightly  curved,  measuring,  according  to 
Fliigge,  about  1  to  2  micra  in  length  by  about  0.3  of  a  micron  in  thickness. 
The  bacilli  are  thus  small  and  slender,  but  are  subject  to  considerable 
variation  from  the  measurements  given,  even  in  one  and  the  same  cul- 
ture. While  ordinarily  single,  the  bacilli  may  be  arranged  end  to  end  in 
short  chains  of  two  and  three.  Longer  chains  may  exceptionally  be 
foTmed  upon  media  which  are  especially  unfavorable  for  its  growth,  such 
as  very  acid  media  or  those  containing  antiseptics. 

Spores  are  not  found.  The  bacilli  are  actively  motile  and  possess 
each  a  single  flagellum  placed  at  one  end. 

Bacillus  pyocyaneus  is  stained  easily  with  all  the  usual  dyes,  but  is 
decolorized  by  Gram's  method.  Irregular  staining  of  the  bacillary  body 
is  common,  but  is  always  an  indication  of  degeneration,  and  not  a 
normal  characteristic,  as,  for  instance,  in  the  diphtheria  group. 

Cultivation. — The  pyocyaneus  bacillus  is  aerobic  and  facultatively 
anaerobic.  It  can  be  adapted  to  absolutely  anaerobic  environments,  but 
does  not  produce  its  characteristic  pigment  without  the  free  access  of 
oxygen.  The  bacillus  grows  readily  upon  the  usual  laboratory  media 
and  is  not  very  sensitive  to  reaction,  growing  equally  well  upon  moder- 
ately alkahne  or  acid  media.  Development  takes  place  at  temperatures 
as  low  as  18°  to  20°  C,  more  rapidly  and  luxuriantly  at  37.5°  C. 

»  Gessard,  These  de  Paris,  1882. 
^Charrin,  "  La  maladie  pyocyanique, "  Paris,  1889. 
677 


578  PATHOGENIC   MICROORGANISMS 

On  agar  slants,  growth  is  abundant  and  confluent,  the  surface  of  the 
agar  being  covered  by  a  moist,  grayish  or  yellowish,  glistening,  even  layer. 
The  pigment  which  begins  to  become  visible  after  about  eighteen  hours 
soon  penetrates  the  agar  itself  and  becomes  diffused  throughout  it, 
giving  the  medium  a  bright  green  fluorescent  appearance,  which  grows 
darker  as  the  age  of  the  culture  increases. 

In  gelatin  stabs,  growth  takes  place  much  more  rapidly  upon  the 
surface  than  in  the  depths.  A  rapid  liquefaction  of  the  gelatin  takes 
place,  causing  a  saucer-shaped  depression.  As  this  deepens,  pigment 
begins  to  form  in  the  upper  layers,  often  visible  as  a  greenish  pellicle. 

In  gelatin  plates,  the  colonies  have  a  characteristic  appearance.  They 
are  round  and  are  composed  of  a  central  dense  zone,  and  a  peripheral, 
loosely  granular  zone,  which  extends  outward  into  the  peripheral  fluidi- 
fied area  in  a  fringe  of  fine  filaments.  When  first  appearing,  they  are 
grayish  yellow,  later  assuming  the  characteristic  greenish  hue. 

In  broth,  growth  is  rapid  and  chiefly  at  the  surface,  forming  a  thick 
pellicle.  Below  this,  there  is  moderate  clouding.  The  pigment  is  formed 
chiefly  at  the  top.  In  old  cultures  there  is  a  heavy  flocculent  precipitate. 
In  fluid  media  containing  albuminous  material,  strong  alkalinity  is 
produced. 

On  potato,  growth  develops  readily  and  a  deep  brownish  pigment  ap- 
pears, which  is  not  unlike  that  produced  by  B.  mallei  upon  the  same 
medium. 

Milk  is  coagulated  by  precipitation  of  casein  and  assumes  a  yellowish- 
green  hue.  In  older  cultures  the  casein  may  again  be  digested  and  liquefied 

The  pigment  of  Bacillus  pyocyaneus  has  been  the  subject  of  much 
investigation.  It  was  shown  by  Charrin  ^  and  others  that  this  pigment 
had  no  relation  to  the  pathogenic  properties  of  the  bacillus.  It  is  found 
in  cultures  as  a  colorless  leukobase  which  assumes  a  green  color  on  the 
addition  of  oxygen.  Conversely,  the  typical  green  "pyocyanin,"  as 
the  pigment  is  caUed,  may  be  decolorized  by  reducing  substances.  This 
explains  the  fact  that  it  is  not  found  in  cultures  sealed  from  the  air.  Pyo-. 
cyanin  may  be  extracted  from  cultures  with  chloroform  and  crystallized 
out  of  such  solution  in  the  form  of  blue  stellate  crystals.  These,  on 
chemical  analysis,  have  been  found  to  belong  to  the  group  of  aromatic 
compounds,  with  a  formula,  according  to  Ledderhose,^  of  Ci4Hi4N20. 

Besides   pyocyanin.   Bacillus   pyocyaneus   produces    another    pig- 


» Charrin,  loc.  cit. 

« Ledderhose,  quoted  from  Boland,  Ceiio.  f.  Bakt.,  xjcv,  1889. 


BACILLUS  PYCXIYANEUS  579 

ment  which  is  fluorescent  and  insoluble  in  chloroform,  but  soluble  in 
water.  ^  This  pigment  is  common  to  other  fluorescent  bacteria,  and  not 
peculiar  to  Bacillus  pyocyaneus.  The  reddish-brown  color  seen  in  old 
cultures^  and  supposed  by  some  writers  to  be  a  third  pigment,  is  probably 
a  derivative  from  pyocyanin  by  chemical  change. 

Chloroform  extraction  of  pyocyanin  from  cultures  may  serve  oc- 
casionally to  distinguish  the  pyocyaneus  bacilli  from  other  similar 
fluorescent  bacteria.  Ernst  has  claimed  that  there  are  two  types  of  B. 
pyocyaneus,  an  a-type  which  produces  only  the  fluorescent,  water- 
soluble  pigment,  and  a  /5-type  which  produces  both  this  and  pyocyanin.^ 

Pathogenicity. — Bacillus  pyocyaneus  is  one  of  the  less  virulent  patho- 
genic bacteria.  It  is  widely  distributed  in  nature  and  may  be  found 
frequently  as  a  harmless  parasite  upon  the  skin  or  in  the  upper  respira- 
tory tracts  of  animals  and  men.  It  has,  however,  occasionally  been 
found  in  connection  with  suppurative  lesions  of  various  parts  of 
the  body,  often  as  a  mere  secondary  invader  in  the  wake  of  another 
incitant,  or  even  as  the  primary  cause  of  the  inflammation.  In  most 
cases  where  true  pyocyaneus  infection  has  taken  place,  the  subject  is 
usually  one  whose  general  condition  and  resistance  are  abnormally  low."* 
Thus  pyocyaneus  may  be  the  cause  of  chronic  otitis  media  in  ill-nour- 
ished children.  It  has  been  cultivated  out  of  the  stools  of  children  suf- 
fering from  diarrhea,  and  has  been  found  at  autopsy  generally  distributed 
throughout  the  organs  of  children  dead  of  gastro-enteritis.^  It  has  been 
cultivated  from  the  spleen  at  autopsy  from  a  case  of  general  sepsis 
following  mastoid  operation.  The  bacillus  has  been  found,  further- 
more, during  life  in  pericardial  exudate  and  in  pus  from  liver  abscesses.® 

Brill  and  Libman,^  as  well  as  Finkelstein,^  have  cultivated 
B.  pyocyaneus  from  the  blood  of  patients  suffering  from  general  sepsis. 
Wassermann  ^  showed  the  bacillus  to  have  been  the  etiological  factor  in 
an  epidemic  of  umbilical  infections  in  new-born  children.  Similar  exam- 
ples of  B.  pyocyaneus  infection  in  human  beings  might  be  enumerated  in 
large  numbers,  and  there  is  no  good  reason  to  doubt  that,  under  given 

•  Boland,  loc.  cit. 

^Gessard,  Ann.  de  Tinst.  Pasteur,  1890,  1891,  and  1892. 
«  Ernst,  Zeit.  f.  Hyg.,  ii,  1887. 
»  Rohner,  Cent.  f.  Bakt.,  xi,  1892. 

•  Neumann,  Jahrb.  f.  Kinderheilk,,  1890. 

•  Kraunhals,  Zeit.  f.  Chir.,  xxxvii,  1893. 

»  Brill  and  Lihman,  Amer.  Jour.  Med.  Sci.,  1899. 

•  Finkelstein,  Cent.  f.  Bakt.,  1899.  f 

•  Wassermann,  Virchow's  Arch.,  clxv,  1901. 


580  PATHOGENIC  MICROORGANISMS 

conditions,  fatal  infections  may  occur.  Such  cases,  however,  are  still  to 
be  regarded  as  depending  more  upon  the  low  resistance  of  the  individual 
attacked  than  upon  the  great  pathogenicity  of  B.  pyocyaneus. 

Many  domestic  animals  are  susceptible  to  experimental  pyocyaneus 
infection,  chief  among  these  being  rabbits,  goats,  mice,  and  guinea- 
pigs.  Guinea-pigs  are  killed  by  this  bacillus  with  especial  ease.  Intra- 
peritoneal inoculation  with  a  loopful  of  a  culture  of  average  virulence 
usually  leads  to  the  death  of  a  young  guinea-pig  within  three  or  four  days. 

Toxins  and  Immunization. — Emmerich  and  Low  have  shown  that 
filtrates  of  old  broth  cultures  of  B.  pyocyaneus  contain  a  ferment-Hke 
substance  which  possesses  the  power  to  destroy  some  other  bacteria, 
apparently  by  lysis.  They  have  called  this  substance  "  pyocyanase  "  and 
claim  that,  with  it,  they  have  succeeded  in  protecting  animals  from 
anthrax  infection.  During  recent  years  pyocyanase  has  been  employed 
locally  for  the  removal  of  diphtheria  bacilli  from  the  throats  of  convales- 
cent cases.  Broth-culture  filtrates  evaporated  to  one-tenth  their  volume 
in  vacuo  are  used  for  this  purpose. 

Pyocyanase  is  exceedingly  thermostable,  resisting  boiling  for  several 
hours,  and  is  probably  not  identical  with  any  of  the  other  toxins  or 
peptonizing  ferments  produced  by  B.  pyocyaneus. 

The  toxins  proper  of  B.  pyocyaneus  have  been  the  subject  of  much 
investigation,  chiefly  by  Wassermann.^  Wassermann  found  that  filtrates 
of  old  cultures  were  far  more  poisonous  for  guinea-pigs  than  extracts 
made  of  dead  bacteria.  He  concludes  from  this  and  other  observations 
that  B.  pyocyaneus  produces  both  an  endotoxin  and  a  soluble  secreted 
toxin.  The  toxin  is  comparatively  thermostable,  resisting  100°  C.  for  a 
short  time.  Animals  actively  immunized  with  living  cultures  of  B.  pyo- 
cyaneus give  rise  in  their  blood  serum  to  bacteriolytic  antibodies  only. 
Immunized  with  filtrates  from  old  cultures,  on  the  other  hand,  their 
serum  will  contain  both  bacteriolytic  and  antitoxic  substances.  The 
true  toxin  of  B.  pyocyaneus  never  approaches  in  strength  that  of  diph- 
theria or  of  tetanus.  Active  immunization  of  animals  must  be  done 
carefully  if  it  is  desired  to  produce  an  immune  serum,  since  repeated 
injections  cause  great  emaciation  and  general  loss  of  strength.  Specific 
agglutinins  have  been  found  in  immune  sera  by  Wassermann^  and 
others.  Eisenberg  ^  claims  that  such  agglutinins  are  active  also  against 
some  of  the  fluorescent  intestinal  bacteria. 


1  Wassermann,  Zeit.  f.  Hyg.,  xxii,  1896.      ^  Wassermann,  Zeit.  f.  Hyg.,  1902. 
^  Eisenberg,  Cent.  f.  Bakt.,  1903. 


BACILLUS  PYOCYANEUS  581 

Bulloch  and  Hunter  *  have  recently  been  able  to  show  that  old 
broth  cultures  of  B.  pyocyaneus  contain  a  substance  capable  of 
hemolyzing  the  red  blood  corpuscles  of  dogs,  rabbits,  and  sheep. 
This  "  pyocyanolysin "  seems  intimately  attached  to  the  bacterial 
body.  Prolonged  heating  of  cultures  does  not  destroy  it.  Heating  of 
hemolytic  filtrates,  however,  destroys  it  in  a  short  time.  The  filtration 
of  young  cultures  yields  very  little  pyocyanolysin  in  the  filtrate.  In 
old  cultures,  however,  a  considerable  amount  passes  into  the  filtrate. 
Whether  or  not  the  hemolytic  power  is  due  to  a  specific  bacterial 
product  or  is  dependent  upon  changes  in  the  culture  fluid,  such  as 
alkalinization,  etc.,  can  not  yet  be  regarded  as  certain. 

Gheorghiewski  ^  claims  to  have  found  a  leucocyte-destroying  ferment 
in  pyocyaneus  cultures. 

>  Bulloch  imd  Hunter,  Cent.  f.  Bakt.,  xxviii,  1900. 
*  Gheorghiewski,  Ann.  de  Tinst.  Pasteur,  xiii,  1899. 


CHAPTER  XLII 
J^IATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM 

(Spirillum  cholerce  asiaticce,  Comrna  Bacillus) 

The  organism  of  Asiatic  cholera  was  unknown  until  1883.  In  this 
year,  Koch/  at  the  head  of  a  commission  established  by  the  German 
government  to  study  the  disease  in  Egypt  and  India,  discovered  the 
"  comma  bacillus  "  in  the  defecations  of  patients,  and  satisfactorily  de- 
termined its  etiological  significance. 

Koch's  investigations  were  carried  out  on  a  large  number  of  cases 
and  many  investigations  have  since  then  corroborated  his  results. 
The  numerous  morphologically  similar  spirilla  which  were  later  found 
in  normal  individuals  and  in  connection  with  other  conditions,  have 
been  shown  by  accurate  bacteriological  methods  to  be  closely  related, 
but  not  identical. 

Apart  from  the"  evidence  of  the  constant  association  of  the  cholera 
vibrio  with  the  disease,  the  etiological  relationship  has  been  clearly 
demonstrated  by  several  accurately  recorded  accidental  infections  oc- 
curring in  bacteriological  workers,  and  by  the  famous  experiment  of 
Pettenkofer  and  Emmerich,  who  purposely  drank  water  containing 
cholera  bacilli.  Both  observers  became  seriously  ill  with  typical  clini- 
cal symptoms  of  cholera,  and  one  of  them  narrowly  escaped  death. 

Morphology  and  Staining. — The  vibrio  or  spirillum  of  cholera  is  a  small 
curved  rod,  varying  from  one  to  two  micra  in  length.  The  degree  of 
curvature  may  vary  from  the  slightly  bent,  comma-like  form  to  a' 
more  or  less  distinct  spiral  with  one  or  twt)  turns.  The  spirals  do  not 
lie  in  the  same  plane,  being  arranged  in  corkscrew  fashion  in  three 
dimensions.  The  spirillum  is  actively  motile  and  owes  its  motility 
to  a  single  polar  flagellum,  best  demonstrated  by  Van  Ermengem's 
flagella  stain.  Spores  are  not  found.  In  young  cultures  the  comma 
shapes  predominate,  in  older  growths  the  longer  forms  are  more  nu- 
merous.   Strains  which  have  been  cultivated  artificially  for  prolonged 

» Koch,  Deut.  med.  Woch.,  1883  and  1884. 

582 


ASIATIC  CHOLERA  AND  THE    CHOLERA  ORGANISM         583 

periods  without  passage  through  the  animal  body  have  a  tendency  to 
lose  the  curve,  assuming  a  more  bacillus-Hke  appearance.  The  spirilla 
are  stained  with  all  the  usual  aqueous  anilin  dyes.  They  are  decolor- 
ized by  Gramas  method.  In  histological  section  they  are  less  easily 
stained,  but  may  be  demonstrated  by  staining  with  alkaline  methylene 
blue. 

Cultivation. — The  cholera  spirillum  grows  easily  upon  all  the  usual 
culture  media,  thriving  upon  meat-extract  as  well  as  upon  meat-infusion 

FiG.  126.— Cholera  Spirillum.     (After  Frankel  and  Pfeiffer.) 

media.  Moderate  alkalinity  of  the  media  is  preferable,  though  slight 
acidity  does  not  prevent  growth. 

In  gelatin  plates  growth  appears  at  room  temperature  within,  twenty- 
four  hours  as  small,  strongly  refracting  yellowish-gray,  pin-head  colonies. 
As  growth  increases  the  gelatin  is  fluidified.  Under  magnification  these 
colonies  appear  coarsely  granular  with  margins  irregular  because  of 
the  liquefaction.  Liquefaction,  too,  causes  a  rapid  development  in 
such  colonies  of  separate  concentric  zones  of  varying  refractive  power. 
Old  strains,  artificially  cultivated  for  long  periods,  lose  much  of  their 
liquefying  power. 

In  gelatin  stab  cultures  fluidification  begins  at  the  surface,  rapidly 
giving^  rise  to  the  familiar  funnel-shaped  excavation. 

Upon  agar  plates,  within  eighteen  to  twenty-four  hours,  grayish, 
opalescent  colonies  appear,  which  are  easUy  differentiated  by  their 


584  PATHOGENIC  MICROORGANISMS 

transparency  from  the  other  bacteria  apt  to  appear  in  feces.  Agar 
plates,  therefore,  are  important  in  the  isolation  of  these  organisms. 

Coagulated  blood  serum  is  fluidified  by  the  cholera  vibrio.  On 
potato,  growth  is  profuse  and  appears  as  a  brownish  coarse  layer.  In 
milk,  growth  is  rapid  and  without  coagulation.  In  broth,  general 
clouding  and  the  formation  of  a  pellicle  result.  The  rapidity  and  luxuri- 
ance of  growth  of  the  cholera  spirillum  upon  alkaline  pepton  solutions 
render  such  solutions  peculiarly  useful  as  enriching  media  in  isolating 
this  microorganism  from  the  stools  of  patients.  In  pepton  solution, 
too,  the  cholera  spirillum  gives  rise  to  abundant  indol,  demonstrated 
in  the  so-called  "cholera-red"  reaction.  This  reaction  has  a  distinct 
diagnostic  value,  but  is  by  no  means  specific.^  In  the  case  of  the  cholera 
vibrio  the  mere  addition  of  strong  sulphuric  acid  suffices  to  bring  out 
the  color  reaction.  This  is  due  to  the  fact  that,  unlike  some  other  indol- 
producing  bacteria,  the  cholera  organism  is  able  to  reduce  the  nitrates 
present  in  the  medium  to  nitrites,  thus  itself  furnishing  the  nitrite 
necessary  for  the  color  reaction.  The  medium  which  is  most  suitable 
for  this  test  is  that  proposed  by  Dunham,^  consisting  of  a  solution 
of  1  per  cent  of  pure  pepton  and  .5  per  cent  NaCl  in  water. 

Dieudonn^^  has  recommended  a  selective  medium  upon  which 
cholera  spirilla  will  grow  well,  but  upon  which  the  colon  bacillus  will 
grow  either  very  sparsely  or  not  at  all.  Its  preparation  is  very  simple. 
To  70  parts  of  ordinary  3  per  cent  agar,  neutralized  to  litmus,  there  are 
added  30  parts  of  a  sterile  mixture  of  defibrinated  beef  blood  and  jiormal 
sodium  hydrate. 

The  latter  is  sterilized  by  steam  before  being  added  to  the  agar. 
This  pure  alkali  agar  is  poured  out  in  plates  and  allowed  to  dry  several 
days  at  37°  or  5  minutes  at  60°.  The  material  to  be  examined  is  smeared 
upon  the  surface  of  these  plates  with  a  glass  rod. 

The  principle  of  this  medium  is  that  cholera  will  grow  in  the  presence 
of  an  amount  of  alkali  which  inhibits  other  fecal  bacteria.  The  medium 
has  been  studied  by  Krimiwiede,  Pratt,  and  Grund,*  who  have  recom- 
mended a  modification.  They  find  the  following  combination  sat- 
isfactory and  an  improvement  upon  Dieudonne's  medium  because 
transparent  and  more  easily  prepared.  They  prepare  the  following 
mixtures : 


*  See  indol  reaction,  p.  167.  ^  Dunham,  Zeit.  f.  Hyg.,  ii,  188;?. 

^  Dieudonni,  A.,  Cent.  Bakt.j  1.,  orig.,  1909. 
*  Krumwiede,  Pratt,  and  Grund,  Jour,  of  Inf.  Dis.,  x,  1912, 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM  585 

Egg-White  Medium. 

A.  White  of  egg  and  water  a.a. 
Sodium  carbonate  cryst.  12  per  cent. 

Mix  in  equal  parts,  steam  in  Arnold  sterilizer  for  20  minutes. 

B.  Meat  pepton  3  per  cent  agar,  neutral  to  litmus. 

30  parts  of  A  are  added  to  70  parts  of  B. 

Another  modification  recommended  by  them  is  as  follows: 

Whole-Egg  Medium. 

A.  Whole  egg  and  water  a.a. 

Sodium  carbonate  12  to  13.5  per  cent. 

Mix  in  equal  parts,  steam  for  20  minutes. 

B.  Meat  free  agar,  viz.,  pepton,  salt,  and  3  per  cent  agar. 

30  parts  of  A  are  mixed  with  70  parts  of  B  while  the  agai*  is  boiling  hot  as 
above. 

The  medium  is  poured  on  the  plates  in  a  thick  layer  and  allowed 
to  stand  open  for  20  to  30  minutes  and  then  the  inoculation  is  carried 
out  by  surface  streaking. 

Isolation. — Isolation  of  the  cholera  vibrio  from  the  feces,  while 
easy  in  many  cases,  is  occasionally  attended  with  some  difficulty 
owing  to  the  large  number  of  other  bacteria  present.  The  most 
satisfactory  method  of  procedure  is  to  inoculate  a  set  of  gelatin 
plates,  another  of  agar  plates,  and  a  number  of  Dunham's  pepton- 
broth  tubes,  with  small  quantities  of  the  suspicious  material.  When 
the  spirilla  are  numerous  they  can  frequently  be  fished  directly  from  sus- 
picious colonies  in  the  plates  and  isolated  for  further  identification. 
When  less  numerous,  they  can  usually  be  found  in  relatively  increased 
numbers  after  eight  or  ten  hours  at  37.5°  C,  in  the  topmost  layers  of 
the  Dunham  broth,  which  is  an  almost  selectively  favorable  medium  for 
these  organisms.  They  collect  at  the  surface  where  free  oxygen  is 
readily  obtained.  From  the  pepton  broth,  plate  dilutions  can  then  be 
prepared  and  colonies  fished.^  Once  isolated,  the  spirilla  are  identified 
by  their  morphology,  by  the  appearance  of  their  colonies,  by  their 
manner  of  growth  upon  gelatin  stabs,  by  the  cholera-red  reaction, 
and,  finally,  by  agglutinative  and  bacteriolytic  tests  in  immune  sera. 
Owing  to  the  existence  of  other  spirilla  morphologically  and  cultu- 
rally similar,  the  serimi  reactions  are  the  only  absolutely  positive  dif- 
ferential criteria. 

1  Abel  und  Claussen,  Cent,  f .  Bakt.,  xvii,  1895. 


586 


PATHOGENIC  MICROORGANISMS 


For  isolation  of  the  bacteria  from  water,  it  is,  of  course,  necessary 
to  use  comparatively  large  quantities.  Fliigge  ^  and  Bitter  advise  the 
distribution  of  about  a  Uter  of  water  in  ten  or  twelve  Erlenmeyer  flasks. 
To  each  of  these  they  add  10  c.c.  of  sterile  pepton-salt  solution  (pepton 
ten  per  cent,  NaCl  five  per  cent).  After  eighteen  hours  at  37.5°  C.  the 
surface  growths  in  these  flasks  are  examined  both  microscopically  and 
culturally  as  before. 

Biological  Considerations. — The  cholera  spirillum  is  aerobic  and 
facultatively  anaerobic.  It  does  not  form  spores.  The  optimum  tem- 
perature for  its  growth  is  about  37.5°  C.  It  grows  easily,  however,  at  a 
temperature  of  22°  C.  and  does  not  cease  to  grow  at  temperatures  as 
high  as  40°.  Frozen  in  ice,  these  bacteria  may  live  for  about  three 
or  four  days.     Roil  ins;  destroys  them  immediately.    A  temperature  of 


h 


I 


Fig.  127. 


Fig.  128. 


Fig.  127.^ — Cholera  Spirillum.     Stab  Culture  in  Gelatin,  three  days  old. 
Fig.  128. — Cholera  Spirillum.     Stab  Culture  in  Gelatin,  six  days  old.     (After 
Frankel  and  Pfeiffer.) 

60°  C.  kills  them  in  an  hour.  In  impure  water,  in  moist  linen,  and  in 
food  stuffs,  they  may  live  for  many  days.  Associated  with  sapro- 
phytes in  feces  and  other  putrefying  material,  and  wherever  active 
acid  formation  is  taking  place,  they  are  destroyed  within  several  days. 
Complete  drying  kills  them  in  a  short  time.  The  common  disin- 
fectants destroy  them  in  weak  solutions  and  after  short  exposures 
(carbolic  acid,  five-tenths  per  cent  in  one-half  hour;  bichlorid  of 
mercury,  1  :  100,000  in  ten  minutes;  mineral  acids,  1  :  5,000  or  10,000 
in  a  few  minutes)  .2 

Pathogenicity. — Cholera  is  essentially  a  disease  of  man.  Endemic  in 
India  and  other  Eastern  countries,  it  has  from  time  to  time  epidemically 
invaded  large  territories  of  Europe  and  Asia,  not  infrequently  assuming 

-^  _  1 /?'%^e,  Zeit.  f.  Hyg.,  xiv,  1893. 

2  Forster,  Hyg.  Rundschau,  1893. 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM  587 

pandemic  proportions  and  sweeping  over  almost  the  entire  earth.^  Five 
separate  cholera  epidemics  of  appalling  magnitude  occurred  during 
the  nineteenth  century  alone;  several  of  these,  spreading  from  India  to 
Asia  Minor,  Egypt,  Russia,  and  the  countries  of  Central  Europe,  reached 
even  to  North  and  South  America.  The  last  great  epidemic  began  about 
1883,  traveled  gradually  westward,  and  in  1892  reached  Germany  where 
it  appeared  with  especial  virulence  in  Hamburg,  and  thence,  fol- 
lowing the  highways  of  ocean  commerce,  entered  America  and  Africa. 
During  this  epidemic  in  Russia  alone  800,000  people  fell  victims  to  the 
disease. 

In  man  the  disease  is  contracted  by  ingestion  of  cholera  organisms 
with  water,  food,  or  any  contaminated  material.  The  disease  is  essen- 
tially an  intestinal  one.  The  bacteria,  very  sensitive  to  an  acid  reaction, 
may  often,  if  in  small  numbers,  be  checked  by  the  normal  gastric  secre- 
tions. Having  once  passed  into  the  intestine,  however,  they  proUferate 
rapidly,  often  completely  outgrowing  the  normal  intestinal  flora.  Fatal 
cases,  at  autopsy,  show  extreme  congestion  of  the  intestinal  walls. 
Occasionally  ecchymosis  and  localized  necrosis  of  the  mucosa  may  be 
present  and  swelling  of  the  solitary  lymph-follicles  and  Peyer's 
patches.  Microscopically  the  cholera  spirilla  may  be  seen  to  have 
penetrated  the  mucosa  and  to  lie  within  its  deepest  layers  close 
to  the  submucosa.  The  most  marked  changes  usually  take  place 
in  the  lower  half  of  the  small  intestine.  The  intestines  are  filled 
with  the  characteristically  fluid,  slightly  bloody,  or  "rice-water" 
stools,  from  which  often  pure  cultures  of  the  cholera  vibrio  can 
be  grown.  The  microorganisms  can  be  cultivated  only  from  the 
intestines  and  their  contents,  and  the  parenchymatous  degenera- 
tions taking  place  in  other  organs  must  be  interpreted  as  being 
purely  of  toxic  origin. 

In  animals,  cholera  never  appears  as  a  spontaneous  disease.  Nikati 
and  Rietsch  ^  have  succeeded  in  producing  a  fatal  disease  in  guinea-pigs 
by  opening  the  peritoneum  and  injecting  cholera  spirilla  directly  into 
the  duodenum.  Koch  ^  succeeded  in  producing  a  fatal  cholera-like 
disease  in  animals  by  introducing  infected  water  into  the  stomach 
through  a  catheter  after  neutralization  of  the  gastric  juice  with  sodium 
carbonate.  At  the  same  time,  he  administered  opium  to  prevent  active 
peristalsis.     A  method  of  infection  more  closely  analogous  to  the  infec- 

^Hirsch,  "Handb.  d.  histor.-geogr.  Path.,"  1881. 

2  Nikati  und  Rietsch,  Deut.  med.  Woch.,  1884; 

3  Koch,  Deut.  med.  Woch.,  1885. 


58§  PATHOGENIC  MICROORGANISMS 

tion  in  man  was  followed  by  Metchnikoff/  who  successfully  produced 
fatal  disease  in  young  suckling  rabbits  by  contaminating  the  maternal 
teat. 

Subcutaneous  inoculation  of  moderate  quantities  of  cholera  spirilla 
into  rabbits  and  guinea-pigs  rarely  produces  more  than  a  temporary 
illness.  Intraperitoneal  inoculation,  if  in  proper  quantities,  generally 
leads  to  death.  It  will  be  remembered  that  when  working  with  intra- 
peritoneal cholera  inoculations  the  phenomenon  of  bacteriolysis  was 
discovered  by  Pfeiffer.^ 

Different  strains  of  cholera  spirilla  vary  greatly  in  their  virulence. 
The  virulence  of  most  of  them,  however,  can  be  enhanced  by  repeated 
passages  through  animals.  Most  of  our  domestic  animals  enjoy  consid- 
erable resistance  against  cholera  infection,  though  under  experimental 
conditions  successful  inoculations  upon  dogs,  cats,  and  mice  have  been 
reported.     Doves  are  entirely  insusceptible.^ 

Hygienic  Considerations.: — The  cholera  spirillum  leaves  the  body  of 
the  infected  subject  with  the  defecations  only.  Infection  takes  place, 
so  far  as  we  know,  only  by  way  of  the  mouth.  From  these  two  facts  it 
follows  that  the  chief  source  of  danger  for  a  community  Ues  in  infection 
of  its  water  supply.  As  a  matter  of  fact  the  bacteria  have  been  fre- 
quently found  in  the  wells,  lakes,  rivers,  and  harbors  of  afflicted  terri- 
tories, and  in  several  cases  it  has  been  possible  to  define  the  Hmits  of 
an  epidemic  almost  precisely  by  the  distribution  of  the  contaminated 
water  supply.  A  classic  example  of  this  is  that  of  the  Hamburg  epi- 
demic, during  which  Altona,  a  town  as  close  to  Hamburg  as  Brooklyn 
is  to  New  York,  with  unrestricted  interurban  traffic  but  with  separate 
water  supply,  was  almost  spared,  while  Hamburg  itself  was  iftidergoing 
one  of  the  most  virulent  epidemics  of  its  history.  It  has  been  statistically 
noted,  moreover,  chiefly  by  Koch,  that  cholera  in  its  spread  not  infre- 
quently follows  the  water  courses.  Apart  from  infection  through  the 
water  supply,  cholera  may  be  transmitted  directly  or  indirectly  by  con- 
tact with  contaminated  linen,  bedclothes,  etc.,  the  organism  being  con- 
veyed to  the  mouth  by  the  fingers,  or  by  infected  food.  Epidemics  due 
to  this  mode  of  infection  alone,  however,  are  apt  to  be  more  narrowly 
localized  and  more  sporadic  in  their  manifestations.  It  is  probable  that 
this  mode  of  infection  is  of  great  importance  in  countries  where  the  disease 

1  Metchnikoff,  Ann.  d.  I'inst.  Pasteur,  1894  and  1896. 

2  pfeiffer,  loc.  cit.  ' 

3  Pfeiffer  und  Nocht,  Zeit.  f .  Hyg.,  vii,  1889. 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM  589 

is  endemic,  but  its  significance  in  producing  epidemics  is  limited  owing  to 
the  fortunately  low  resistance  of  the  spirillum  to  desiccation.  The 
sudden  appearance  of  cholera  in  a  place  far  distant  from  the  seat  of  a 
prevalent  epidemic  may  be  explained  by  the  occasional  presence  of 
cholera  spirilla  in  the  dejecta  of  convalescents  as  late  as  two  or  three 
weeks  after  apparent  recovery  from  the  disease  and  consequent  release 
from  quarantine. 

Cholera  Toxin. — ^The  absence  of  the  cholera  spirilla  from  the  in- 
ternal organs  of  fatal  cases,  in  spite  of  the  severe  general  symptoms 
of  the  disease,  points  distinctly  to  the  existence  of  a  strong  poison  pro- 
duced in  the  intestine  by  the  microorganisms  and  absorbed  by  the 
patient.  It  was  in  this  sense,  indeed,  that  Koch  first  interpreted  the 
clinical  picture  of  cholera.  Numerous  investigations  into  the  nature  of 
these  toxins  have  been  made,  the  earUer  ones  defective  in  that  definite 
identification  of  the  cultures  used  for  experimentation  were  not  carried 
out. 

Pfeiffer,Vin  1892,  was  able  to  show  that  filtrates  of  young  bouillon 
cultures  of  cholera  spirilla  were  but  sHghtly  toxic,  whereas  the  dead 
bodies  of  carefully  killed  agar  cultures  were  fatal  to  guinea-pigs  even  in 
small  quantities.  In  consequence,  he  regarded  the  cholera  poison  as 
consisting  chiefly  of  an  endotoxin.^  The  opinion  as  to  the  endotoxic 
nature  of  the  cholera  poison  is  not,  however,  shared  by  all  workers. 
Metchnikoff,  Roux,  and  Salimbeni,^  in  1896,  succeeded  in  producing 
death  in  guinea-pigs  by  introduction  into  their  peritoneal  cavities  of 
cholera  cultures  enclosed  in  celloidin  sacs.  Brau  and  Denier,^  and, 
more  recently,  Kraus,^  claim  that  they  have  succeeded  not  only  in 
demonstrating  a  soluble  toxin  in  alkaline  broth  cultures  of  cholera  - 
spirilla,  but  in  producing  true  antitoxins  by  immunization  with  such 
cultures.  It  appears,  therefore,  that  the  poisonous  action  of  the  cholera 
organisms  may  depend  both  upon  the  formation  of  true  secretory  toxins 
and  upon  endotoxins.  Which  of  these  is  paramount  in  the  produc- 
tion of  the  disease  can  not  be  at  present  definitely  stated.  In  favor 
of  the  great  importance  of  the  endotoxic  elements  is  the  failure, 
thus  far,  to  obtain  successful  therapeutic  results  with  supposedly 
antitoxic  sera. 

1  Pfeiffer,  Zeit.  f.  Hyg.,  xi,  1892. 

2  Pfeiffer  und  Wassermann,  Zeit.  f.  Hyg.,  xiv,  1893. 

3  Metchnikoff,  Roux,  et  Salimheni,  Ann.  d.e  I'inst.  Pasteur,  1896. 
^  Brau  et  Denier,  Comptes  rend,  de  I'acad.  des  sci.,  1906. 

6  R,  Kraus,  Cent,  f .  Bakt.,  1906. 


590  PATHOGENIC  MICROORGANISMS 

Cholera  Immunization. — One  attack  of  cholera  confers  protection 
against  subsequent  infection.  Active  immunization  of  animals  may 
be  accomplished  by  inoculation  of  dead  cultures,  or  of  small  doses  of 
living  bacteria.  In  the  serum  of  immunized  animals  specific  bacterio- 
lytic and  agglutinating  substances  are  found.  The  discovery  of  bacte- 
riolytic immune  bodies,  in  fact,  was  made  by  means  of  cholera  spirilla. 
Both  the  bacteriolysins  and  the  agglutinins,  because  of  their  specificity, 
are  of  great  importance  in  making  a  bacteriological  diagnosis  of  true 
cholera  organisms. 

Protective  inoculation  of  man  has  been  variously  attempted  by 
Ferran  ^  and  others.  Experiments  on  a  large  scale  were  done,  more  re- 
cently, by  Haffkine,^  who  succeeded  in  producing  an  apparently  dis- 
tinct prophylactic  immunization  by  the  subcutaneous  inoculation  of 
dead  cholera  cultures.  Similar  immunization  with,  bacterial  filtrates 
has  been  attempted  by  Bertarelli.^  Strong  reconunends  immunization 
with  filtrates  from  autolyzed  cultures. 

CHOLERA-LIKE  SPIRILLA 

The  biological  group  of  the  vibriones,  to  which  the  cholera  spirillum 
belongs,  is  a  large  one,  numbering  probably  over  a  hundred  separate 
species.  Most  of  these  are  of  bacteriological  importance  chiefly  because 
of  the  difficulties  which  they  add  to  the  task  of  differentiation,  for  while 
some  of  them  simply  bear  a  morphological  resemblance  to  the  true 
cholera  vibrio,  others  can  be  distinguished  only  by  their  serum  reac- 
tions and  pathogenicity  for  various  animals.  Additional  difficulty, 
too,  is  contributed  by  the  fact  that  within  the  group  of  true  cholera 
organisms  occasional  variations  in  agglutinability  and  bacteriolytic 
reactions  may  exist.  Certain  strains,  too,  the  six  El  Tor  cultures 
isolated  by  Gottschlich,  while  in  every  respect  similar  to  true  cholera 
spirilla,  are  considered  as  a  separate  sub-species  by  Kraus,^  because  of 
their  ability  to  produce  hemolytic  substances,  a  function  lacking  in 
other  cholera  strains. 

Spirillum  Metchnikovi. — ^This  spirillum  was  discovered  by  Gamaleia  ^ 
in  the  feces  and  blood  of  domestic  fowl,  in  which  it  had  caused  an  in- 
testinal disease.    Morphologically  and  in  staining  reactions  it  is  identical 

1  Ferran,  Comptes  rend,  de  I'acad.  des  sciences,  1885. 

2  Haffkine,  BuU.  med.,  1892.  ^  Bertarelli,  Deut.  med.  Woch.,  33,  1904, 
^Kraus,  Kraus  and  Levaditi;  "Handbuch,"  vol.  i,  p.  186. 

^  Gamaleia,  Ann,  de  Tinst.  Pasteur,  1883. 


ASIATIC  CHOLERA  AND  THE  CHOLERA  ORGANISM  591 

with  Spirillum  cholerse  asiaticse.  It  possesses  a  single  polar  flagellum, 
and  is  actively  motile.  Culturally  it  is  identical  with  Vibrio  cholerae 
except  for  slightly  more  luxuriant  growth  and  more  rapid  fluidification 
of  gelatin.     It  gives  the  cholera-red  reaction  in  pepton  media. 

It  is  differentiated  from  the  cholera  vibrio  by  its  power  to  produce 
a  rapidly  fatal  septicemia  in  pigeons  after  subcutaneous  inoculation  of 
minute  quantities.^  It  is  much  more  pathogenic  for  guinea-pigs  than 
the  cholera  vibrio.  It  is  not  subject  to  lysis  or  agglutinated  by  cholera 
immune  sera. 

Spirillum  Massaua. — This  organism  was  isolated  at  Massaua  by 
Pasquale  ^  in  1891  from  the  feces  of  a  clinically  doubtful  case  of  cholera. 
Culturally  and  morphologically  it  is  much  like  the  true  cholera  vibrio, 
but  in  pathogenicity  is  closer  to  Spirillum  Metchnikovi,  in  that  small 
quantities  produce  septicemia  in  birds.  It  possesses  four  flagella. 
It  does  not  give  a  specific  serimi  reaction  with  cholera  immune 
serum. 

Spirillum  of  Finkler-Prior.^ — Isolated  by  Finkler  and  Prior  from  the 
feces  of  a  case  of  cholera  nostras.  Morphologically  it  is  like  the  true 
cholera  spirillum,  though  slightly  larger  and  less  uniformly  curved. 
Culturally  it  is  much  like  the  cholera  vibrio,  but  grows  more  rapidly 
and  thickly  upon  the  usual  media.  It  does  not  give  the  cholera-red 
reaction,  nor  does  it  give  specific  serum  reactions  with  cholera  im- 
mune serum. 

•  Spirillum  Deneke.'* — ^A  vibrio  isolated  by  Deneke  from  butter.  Much 
like  that  of  Finkler-Prior.     It  does  not  give  the  cholera-red  reaction. 

1  Pfeiffer  und  Nocht,  Zeit.  f .  Hyg.,  vii,  1889. 

2  Pasquale,  Giorn.  med.  de  r.  eserc.  ed.  R.  Marina,  Roma,  1891. 
'  Finkler  und  Pricyr,  Erganz.  Hefte,  Cent.  f.  allg.  ges.  Phys.,  1884. 
*  Deneke,  Deut.  med.  Woch.,  iii,  1885. 


^ 


CHAPTER  XLIII 
DISEASES   CAUSED    BY    SPIROCHETES  (TREPONEMATA) 

The  microorganisms  known  as  spirochsetes  are  slender,  undulatingj 
corkscrew-like  threads  which  show  definite  variations  both  structurally 
and  culturally  from  the  bacteria  as  a  class.  Most  important  among 
them  are  the  spirochsete  of  relapsing  fever,  Spirochaete  pallida  of 
syphilis,  the  spirillum  of  Vincent,  Spirochsete  refringens.  Spirillum 
gallinarum,  a  microorganism  which  causes  disease  in  chickens, 
Spirochaete  anserina,  which  causes  a  similar  condition  in  geese,  and 
several  species  which  have  been  found  as  parasites,  both  in  animal ° 
and  in  man,  without  having  definite  etiological  connection  with  disease. 

The  classification  of  these  various  species  in  one  group  is  rather  more 
a  matter  of  convenience  than  one  of  scientific  accuracy,  since  our  knowl- 
edge of  them  is  not  far  advanced,  and  our  inability  to  cultivate  almost 
all  of  them  has  not  permitted  their  detailed  biological  study.  Formerly 
many  of  these  organisms  were  regarded  as  bacteria  belonging  to  the  gen- 
eral group  of  the  spirilla.  Recently  Schaudinn,^  the  discoverer  of  the 
syphilis  spirochaete,  has  claimed,  upon  the  basis  of  a  careful  morphological 
study,  that  many  of  these  forms  are  actually  protozoa.  He  based  this 
claim  upon  the  observation  that  stained  preparations  often  showed  undu- 
lating membranes  extending  along  the  long  axis  of  the  microorganisms 
and  that  definite  nuclear  structures  were  demonstrable.  This  observer 
also  claimed  that  many  of  the  spiral  forms  reproduce  by  cleavage  along 
the  longitudinal  axis.  Other  observers  have  not  agreed  with  this  view, 
Laveran,^  Novy  and  Knapp,^  and  others  assertiifg  that  their  own  obser- 
vations indicate  a  close  relationship  of  these  microorganisms  to  the  true 
bacteria.  Whatever  the  final  conclusion  may  be,  the  question  is  more 
or  less  an  academic  one,  in  that  our  ideas  as  to  the  exact  line  of  division 
between  the  unicellular  animals  and  the  unicellular  plants  is  not  by  any 
means  founded  upon  a  sound  basis.     In  common  with  the  bacteria,  most 

*  Schaudinn,  Arb.  a.  d.  kais.  Gesundheitsamt,  1904.   . 
^Laveran,  Comptes  rend,  de  Tacad.  des  sci.,  1902  and  1903. 
3  Novy  and  Knapp,  Jour,  of  Infec.  Dis.,  3,  1906. 
592 


DISEASES  CAUSED  BY  SPIROCILETES  593 

of  these  microorganisms  have  the  power  of  .multipHcation  by  transverse 
fission.  They  possess  flagella  and,  in  the  case  of  some  of  them  at  least, 
definite  immune  bodies  can  be  demonstrated  in  the  serum  of  infected 
subjects  similar  to  those  produced  by  bacteria  during  infection.  The 
undulating  membranes  and  the  definite  differentiation  between  nucleus 
and  cytoplasm  claimed  for  them  by  some  observers  have  not  been  uni- 
formly confirmed,  and  their  similarity  to  the  trypanosomes  has  not 
therefore  been  established.  On  the  other  hand,  none  of  these  micro- 
organisms has  so  far  been  successfully  cultivated  upon  artificial  media, 
with  the  exception  of  the  spirilla  which  occur  in  Vincent's  angina.  For 
some  of  the  diseases  caused  by  this  class  of  parasites,  moreover,  trans- 
mission by  an  intermediate  host,  in  which  the  spirilla  undergo  multipli- 
cation, has  been  definitely  shown,  a  fact  which  corresponds  with  the 
conditions  observed  in  many  protozoan  infections.  Upon  a  careful  re- 
view of  these  various  data,  it  seems  to  be  fully  justified,  on  the  basis 
of  our  present  knowledge,  to  group  these  microorganisms,  as  KoUe  and 
Hetsch  1  have  done,  in  a  class  between  bacteria  and  protozoa. 

The  terms  spirochsete  and  spirillum  have  been  indiscriminately  used. 
In  the  original  classification  of  Migula  the  difference  between  the  two 
groups  was  based  upon  the  rigidity  of  the  cell  body  in  the  case  of  the 
spirilla  and  the  sinuous  or  flexible  nature  of  the  cell  in  the  case  of  the 
spirochaetse.  Although  the  term  spirillum  is  still  colloquially  used  for 
some  members  of  this  group,  merely  because  of  past  usage,  it  would  be 
better  to  speak  of  all  the  microorganisms  here  grouped  together  by  the 
term  ''spirochsetes." 

SYPHILIS  AND  SPIROCUffiTA  PALLIDA 

{Treponema  pallidum) 

The  peculiar  manifestations  of  syphilis,  its  mode  of  transmission, 
and  the  fact  that  its  primary  lesion  was  always  located  at  the  point 
of  contact  with  a  preceding  case,  have  always  stamped  it  as  unques- 
tionably infectious  in  nature.  Until  very  recently  the  microorgan- 
ism which  gives  rise  to  syphilis  was  unknown.  Many  bacteriologists 
had  attacked  the  problem  and  many  microorganisms  for  which  defi- 
nite etiological  importance  was  claimed  had  been  described.  Most  of 
these  announcements,  however,  aroused  little  more  than  a  sensational 
interest  and  received  no  satisfactory  confirmation.     A  bacillus  described 

^KolU  und  Hetsch,  "Die  experimentelle  Bakt.,"  Berlin,  1906. 


594  PATHOGENIC  MICROORGANISMS 

by  Lustgarten  ^  in  1884  seemed,  for  a  time,  to  have  solved  the  mystery. 
The  Lustgarten  bacillus  was  an  acid-fast  organism  very  similar  to 
Bacillus  tuberculosis,  and  found  by  its  discoverer  in  a  large  num- 
ber of  syphilitic  lesions.  The  observation,  at  first,  aroused  much  interest 
and  received  some  confirmation.  Later  extensive  investigations,  how- 
ever, failed  to  uphold  the  etiological  relationship  of  this  bacillus  to  the 
disease  and  practically  identified  it  with  the  smegma  bacillus,  so  often 
a  saprophyte  upon  the  mucous  membranes  of  the  normal  genitals. 

In  1905,  Schaudinn,2  a  German  zoologist,  working  in  collaboration 
with  Hoffmann,  investigated  a  number  of  primary  syphilitic  indurations 
and  secondarily  enlarged  lymph  nodes,  and  in  both  lesions  discovered  a 
spirochaete  similar  to,  but  easily  distinguished  from,  the  spirochaetes 


Fig.   129. — ^SriROCHiETA  pallida.     Smear  preparation  from  chancre  stained 
by  -the  india-ink  method. 

already  known.     He  failed  to  find  similar  microorganisms  in  uninfected 
human  beings. 

The  microorganism  described  by  him  as  "Spirochseta  palUda"  is  an 
extremely  delicate  undulating  filament  measuring  from  four  to  ten  micra 
in  length,  with  an  average  of  seven  micra,  and  varying  in  thickness  from 
an  immeasurable  delicacy  to  about  0.5  of  a  micron.  It  is  thus  distinctly 
smaller  and  more  delicate  than  the  spirochaete  of  relapsing  fever.  Ex- 
amined in  fresh  preparations  it  is  distinctly  motile,  its  movements  con- 
sisting in  a  rotation  about  the  long  axis,  gliding  movements  backward 
and  forward,  and,  occasionally,  a  bending  of  the  whole  body.  Its  con- 
volutions, as  counted  by  Schaudinn,  vary  from  3  to  12  and  differ  from 
those  observed  in  many  other  spirochaetes  by  being  extremely  steep,  or, 
in  other  words,  by  forming  acute,  rather  than  obtuse,  angles.  The  ends 
of  the  microorganism  are  delicately  tapering  and  come  to  a  point.     In 

^  Lustgarten,  Wien.  med.  Woch.,  xxxiv,  1884. 

2  Schaudinn  und  Hoffmann,  Arb.  a.  d.  kais.  Gesundheitsamt,  22,  1905. 


DISEASES  CAUSED  BY  SPIROCHETES  595 

his  first  investigations,  Schaudinn  was  unable  to  discover  flagella  and 
believed  that  he  saw  a  marginal  undulating  membrane  similar  to  that 
noticed  in  the  trypanosomes.  Later  observations  by  this  observer,  as 
well  as  by  others,  revealed  a  delicate  flagellum  at  each  end,  but  left  the 
existence  of  an  undulating  membrane  in  doubt.  Uncertain,  in  his  later 
investigations,  whether  the,  microorganisms  described  by  him  could 
scientifically  be  classified  with  the  spirochsete  proper,  Schaudinn  sug- 
gested the  name  of  "  Treponema  pallidum.^' 

In  the  same  preparations  in  which  Spirochaeta  pallida  was  first 
seen,  other  spirochetes  were  present,  which  were  easily  distinguished 
from  the  former  by  their  coarser  contours,  their  flatter  and  fewer  undula- 
tions, their  more  highly  refractile  cell  bodies,  and,  in  stained  prepara- 
tions, their  deeper  color.  These  microorganisms  were  not  found 
regularly,  and  were  interpreted  merely  as  fortuitous  and  unimportant 
companions.  To  them  Schaudinn  gave  the  name  of  "  Spirochseta  re- 
fringens." 

The  epoch-making  discovery  of  Schaudinn  and  Hoffmann  was  soon 
confirmed  by  many  observers,  and  the  etiological  relationship  of  Spiro- 
chaeta pallida  to  syphilis  may  now  be  regarded  as  an  accepted  fact. 
Although  our  inability  to  cultivate  the  microorganism  has  made  it 
impossible  to  carry  out  Koch's  postulates,  nevertheless  indirect  evi- 
dence of  such  a  convincing  nature  has  accumulated  that  no  reasonable 
doubt  as  to  its  caustive  importance  can  be  retained.  The  spirochaetes 
have  been  found  constantly  present  in  the  primary  and  secondary 
lesions  of  all  carefully  investigated  cases,  and,  so  far,  have  invariably 
been  absent  in  subjects  not  afflicted  with  syphilis. 

Schaudinn  himself,  not  long  after  his  original  communication,  was. 
able  to  report  seventy  cases  of  primary  and  secondary  syphilis  in  which 
these  microorganisms  were  found.  Spitzer^  found  them  constantly 
present  in  a  large  number  of  similar  cases.  Sobernheim  and  Tomas- 
czewski^  found  the  spirochaetes  in  fifty  cases  of  primary  and  secondary 
S3rphilis,  but  failed  to  find  them  in  eight  tertiary  cases.  Mulzer,^  who 
found  the  microorganisms  invariably  in  twenty  cases  of  clinical  syphiHs, 
failed  to  find  them  in  fifty-six  carefully  investigated  non-syphilitic  sub- 
jects. The  voluminous  confirmatory  literature  which  has  accumulated 
upon  the  subject  can  not  here  be  reviewed.  The  presence  of  these 
spirochaetes  in  the  blood  at  certain  stages  of  the  disease  has  been  demon- 

1  S-pitzer,  Wien.  klin.  Woch.,  1905. 

2  Sobernheim  und  Tomasczewski,  Miinch.  med.  Woch.,  1905. 

'  MtUzer,  Berl.  klin.  Woch.,  1905,  and  Archiv  f .  Dermat.  u.  Syph.,  79,  1906. 


596  PATHOGENIC  MICROORGANISMS 

strated  by  Bandi  and  Simonelli^  who  found  them  in  the  blood  taken 
from  the  roseola  spots,  and  by  Levaditi  and  Petresco  ^  who  found  them 
in  the  fluid  of  blisters  produced  upon  the  skin. 

In  tertiary  lesions  the  spirochsetes  have  been  found  less  regularly 
than  in  the  primary  and  secondary  lesions,  but  positive  evidence  of  their 
presence  has  been  brought  by  Tomasczewsjd,^  Ewing,^  and  others  who 
succeeded  in  demonstrating  them  in  gummata.  Noguchi  and  Moore  ^ 
have  recently  found  the  Spirochseta  pallida  in  the  brain  of  patients  dead 
of  general  paresis. 

In  congenital  syphilis,  many  observers,  have  found  Spirochseta 
pallida  in  the  lungs,  liver,  spleen,  pancreas,  and  kidneys,  and,  in  isolated 
cases,  in  the  heart  muscle.  The  organisms  were  always  present  in  large 
numbers  and  practically  in  pure  culture.  These  results  more  than  any 
others  seem  to  furnish  positive  proof  of  the  etiological  relationship  be- 
tween the  spirochsete  and  the  disease. 

Demonstration  of  Treponema  pallidum. — In  the  living  state  the 
spirochsetes  have  been  observed  in  the  hanging  drop  or  under  a  cover- 
slip  rimmed  with  vaseline.  It  is  extremely  important,  in  preparing  such 
specimens  from  primary  lesions  or  from  lymph  glands,  to  obtain  the 
material  from  the  deeper  tissues,  and  thus  as  uncontaminated  as  possible 
by  the  secondary  infecting  agents  present  upon  the  surface  of  an  ulcer, 
and  also  as  free  from  blood  as  possible.  An  ordinary  microscope 
and  condenser  may  be  used,  provided  that  the  light  is  cut  down  con- 
siderably by  means  of  the  iris  diaphragm.  This  method  is,  however, 
difficult  and  uncertain.  It  is  better  to  employ  a  special  device  known 
as  a  "condenser  for  dark-field  illumination''  (Dunkel-Kammer- 
Beleuchtung).  This  apparatus  is  screwed  into  the  place  of  the  Abbe 
condenser.  The  preparation  is  made  upon  a  slide  and  covered  with  a 
cover-slip  as  usual.  A  drop  of  oil  is  then  placed  upon  the  upper  sur- 
face of  the  dark  chamber  and  the  slide  laid  upon  it  so  that  an  even 
layer  of  oil,  without  air-bubbles,  intervenes  between  the  top  of  the  dark 
chamber  and  the  bottom  of  the  slide.  The  preparation  is  then  best 
examined  with  a  high-power  dry  lens.  An  arc  light  furnishes  the  most 
favorable  illimoination.     In  such  preparations  the  highly  refractive  cell- 


1  Bandi  und  SirrumeUi,  Cent,  f .  Bakt.,  40,  1905. 

*  Levaditi  et  Petresco,  Presse  m6d.,  1905. 

'  Tomasczewski,  Miinch.  med.  Woch.,  1906. 

*  Swing,  Proc.  N.  Y.  Path.  Soc,  N.  S.,  5,  1905.  \ 

*  Noguchi  and  Moore,  Jour.  Exp.  Med.,  xvii,  1913.^ 


DISEASES  CAUSED  BY  SPIROCHiETES  597 

bodies  stand  out  against  the  black  background,  and  the  motility  of  the 
organisms  may  be  observed.^ 

The  dark-field  condenser  is  without  question  the  easiest  method  of 
finding  the  Spirochseta  pallida.  Its  use  is  easily  learned  and  the  appara- 
tus is  sufficiently  cheap  so  that  it  lends  itself  to  the  use  of  the  clinic  and 
the  office.  With  very  little  practice  it  is  possible  to  detect  the  spiro- 
chsete  in  suspension  if  care  is  taken  that  not  too  much  blood  or  other 
solid  particles  are  mixed  with  the  preparation.  Should  it  be  impossible 
to  obtain  the  material  scraped  from  syphilitic  lesions  in  a  sufficiently 
dilute  condition  it  is  best  to  emulsify  it  in  a  drop  or  two  of  human 
ascitic  fluid. 

Examination  in  Smears. — The  Spirochseta  pallida  can  not  be 
stained  with  the  weaker  anilin  dyes,  and  even  more  powerful  dyes,  such 
as  carbol-fuchsin  and  gentian-violet,  give  but  a  pale  and  unsatisfactory 
preparation.  ,  The  staining  method  most  commonly  used  is  the  one 
originally  recommended  by  Schaudinn  and  Hoffmann.  This  depends 
upon  the  use  of  Qiemsa's  azur-eosin  stain  employed  in  various  modi- 
fications. The  most  satisfactory  method  of  applying  this  solution  is 
as  follows: 

Make  smears  upon  slides  or  cover-slips,  if  possible  from  the  depth  of  the 
lesions,  as  free  as  possible  from  blood. 

Fix  in  methyl  alcohol  for  ten  to  twenty  minutes  and  dry. 

Cover  the  preparation  with  a  solution  freshly  prepared  as  follows: 

Distilled  water 10  c.c. 

Potassium  carbonate  1  : 1,000 5-10  gtt. 

Add  to  this: 

Giemsa's  solution  {Jilr  Romanowski  Fdrbung) 10-12  gtt. 

This  staining  fluid  is  left  on  for  one  to  four  hours,  preferably  in  a  moist 
chamber.    Wash  in  running  water.    Blot. 

By  this  method  Spirochseta  pallida  is  stained  characteristically  with 
a  violet  or  reddish  tinge. 

A  rapid  and  convenient  method  for  staining  such  smears  consists  in 
the  use  of  azur  I  and  eosin  in  aqueous  solutions  as  recommended  by 
Wood  (see  section  on  Staining,  page  109).  The  smears  are  fixed  in 
methyl  alcohol  as  before  and  are  then  flooded  with  the  azur  I  solution. 
The  eosin  solution  is  then  dropped  on  the  preparation  until  an  iridescent 

1  For  a  critical  summary  of  the  various  methods  of  dark-field  illumination,  the 
reader  is  referred  to  an  article  by  Siedentopf,  Zeit.  f.  wiss.  Mikrosc,  xxv,  1908. 


598  PATHOGENIC  MICROORGANISMS 

pellicle  begins  to  form.    Satisfactory  preparations  may  be  obtained  by 
this  method  after  ten  or  fifteen  minutes  of  staining. 

An  excellent  method  of  staining  the  treponema  pallidum  in  smear- 
preparations  is  that  of  Fontana.^  For  this  method,  the  following  solu- 
tions are  necessary: 

1 .  Acetic  acid 1  c.c. 

Formalin 2  c.c. 

Distilled  water 100  c.c. 

Leave  in  one  minute;  wash  in  water. 

2.  Phenol  86%  (Uquefied  crystals) 1  c.c. 

Tannic  acid 5  grams 

Distilled  water 83  c.c. 

Cover  preparation  with  this  and  steam  gently  one-half  minute;  wash. 

3 .  Silver  nitrate 0 .  25  grams 

Distilled  water 100  c.c. 

Ammonia  q.  s. 
Add  ammonia  drop  by  drop  until  the  precipitate  which  first  appears  goes 
into  solution.     Steam  one-half  minute;  wash. 

Recently  a  rapid  and  extremely  simple  and  reliable  method  for  the 
demonstration  of  Spirochseta  pallida  in  smears,  by  the  use  of  India  ink, 
has  been  described. 

Smears  are  prepared  in  the  following  way:  A  drop  of  the  fluid 
squeezed  out  of  the  syphilitic  lesion,  as  free  as  possible  from  blood  cells, 
is  mixed,  on  a  slide,  with  a  drop  of  India  ink  (best  variety  is  '' Chin  chin" 
Giinther-Wagner  Liquid  Pearl  ink),  and  the  mixture  smeared  with  the 
edge  of  another  slide  as  in  making  blood  smears.  When  the  smear  dries, 
which  takes  about  a  minute,  it^may  be  immediately  examined  with  an 
oil-immersion  lens.  The  organisms  are  seen  unstained  on  a  black  back- 
ground.    (See  Fig.  129,  p.  594.) 

Demonstration  of  Spirochetes  in  Tissues. — Ordinary  histo- 
logical staining  methods  do  not  reveal  the  spirochaetes  in  tissue  sections. 
It  is  customary,  therefore,  to  employ  some  modification  of  Cajal's 
silver  impregnation.  The  technique  most  commonly  employed  is  that 
known  as  Levaditi's  method,'^  which  is  carried  out  as  follows: 

The  fresh  tissue  is  cut  into  small  pieces  which  should  not  be  thicker  than 
2  to  4  millimeters. 

Fix  in  10%  formalin  (4%  formaldehyde)  for  twenty-four  hours.  Wash  in 
water. 

^See  Levaditi  and  Bankowski:  Ann.  de  I'lnsti.  Past.,  1913,  XXVII,  p.  583. 
2  Levaditi,  Comptes  rend,  de  la  see.  de  bioL,  59,  1905. 


DISEASES  CAUSED  BY  SPIROCHiETES  599 

Dehydrate  in  96%  alcohol  twenty-four  hours.    Wash  in  water. 
Place  in  a  3%  silver-nitrate  solution  at  incubator  temperature  (37.5°  C.) 
and  in  the  dark  for  3  to  5  days.    Wash  in  water  for  a  short  time. 
Place  in  the  following  solution  (freshly  prepared) : 

PjTogallic  acid 2-4  grams. 

Formalin 5  c.c. 

Distilled  water 100    " 

Leave  in  this  for  twenty-four  to  forty-eight  hours  at  room  temperature. 
Wash  in  water. 
Dehydrate  in  graded  alcohols. 
Embed  in  paraffin  and  cut  thin  sections. 

The  sections  may  be  examined  without  further  staining,  or,  if  desired, 
may  be  weakly  counterstained  with  Giemsa's  solution  or  hematoxyUn. 

A  modification  of  this  method  which  has  been  much  recommended  is 
that  of  Levaditi  and  Manouelian}  The  directions  given  by  these  authors 
are  as  follows: 

Fix  in  formahn  as  in  previous  method. 

Dehydrate  in  96%  alcohol  12  to  24  hours.    Wash  in  distilled  water. 

Place  in  a  1%  silver-nitrate  solution  to  which  10%  of  pyridin  has  been  added 
just  before  use. 

Leave  in  this  solution  for  2  to  3  hours  at  room  temperature  and  from  four 
to  six  hours  at  50°  C.  approximately. 

Wash  rapidly  in  10%  pyridin. 

Place  in  a  solution  containing  4%  of  pyrogallic  acid  to  which  10%  of  C.  P. 
acetone,  and  15%  (per  volume)  of  pyridin  have  been  added  just  before  use. 
Leave  in  this  solution  2  to  3  hours. 

Wash  in  water,  dehydrate  in  graded  alcohols,  and  embed  in  paraffin  by  the 
usual  technique. 

Examined  after  treatment  by  either  of  these  methods,  the  spiro- 
chaetes  appear  as  black,  untransparent  bodies  lying  chiefly  extracellu- 
larly.  They  are  characteristically  massed  about  the  blood-vessels  of  the 
organs  and  only  exceptionally  seem  to  penetrate  into  the  interior  of 
the  parenchyma  cells. 

Attempts  at  cultivating  Spirochseta  pallida  were  at  first  unsuccessful. 
Recently  Schereschewsky  ^  has  reported  that  he  has  succeeded  in  ob- 
taining multiplication  of  the  organisms  on  artificial  media  as  follows: 
Sterile  horse  serum  in  centrifuge  tubes  was  coagulated  at  60°  C.  until 
it  assumed  a  jelly-like  consistency.    It  was  then  placed  in  the  incubator 

^  Levaditi  et  Manoiielian,  Comptes  rend,  de  la  see.  de  bid.,  60,  1906. 
2  Schereschewsky,  Deut.  med.  Woch.,  N.  S.,  xix  and  xxix,  1909. 


600  «        PATHOGENIC  MICROORGANISMS 

at  37.5°  C.  for  three  days  before  being  used.  The  cultures  were  planted 
by  snipping  off  a  small  piece  of  tissue  from  a  sj^hilitic  lesion,  dropping 
it  into  such  a  tube,  and  Causing  it  to  sink  to  the  bottom  by  means  of 
centrifugalization.  The  tube  was  then  tightly  stoppered  with  a  cork. 
In  such  anaerobic  serum  cultures  Schereschewsky  claims  to  have  grown 

the   organisms   for    several    generations, 
though  not  in  pure  culture. 

Miihlens  also  obtained  growth  of 
Spirochseta  pallida  in  horse  serum  agar 
by  a  method  which  is  very  similar  to 
that  of  Schereschewsky.  The  most  ex- 
tensive and  convincing  work  on  trepon- 
ema  pallidum  has  been  done  recently  by 
Noguchi.     Noguchi  ^  began  his  work  in 

Fig.  130. — SpIROCH.ETA    pal-        mirk         ^    inii         XS'      n     j.  r    1  1 

CI  .1       u       lyiO  and  1911.     His  first  successful  cul- 

LiDA.  Spleen,  congenital  syph- 
ilis. (Levaditi  method.)  tivations  were  made  from  the  syphilis- 
infected  testicles  of  rabbits,  and  after 
many  unsuccessful  attempts,  with  slightly  varying  media  and 
technique,  he  finally  succeeded  in  the  following  way:  He  pre- 
pared tubes  (20  cm.  high  and  1.5  cm.  wide),  containing  10  c.c. 
of  a  serum-water  made  of  distilled  water,  three  parts;  and  horse, 
sheep,  or  rabbit  serum,  one  part.  These  were  sterilized  by  the 
fractional  method  in  the  usual  way  (15  minutes  each  day).  Into  them 
was  then  placed  a  small  piece  of  sterile  rabbit  kidney  or  testicle  and  a 
bit  of  the  testicle  of  a  syphilitic  rabbit,  in  which  many  spirochaetes  were 
present.  The  fluid  was  then  covered  with  sterile  paraffin  oil  and  placed 
in  an  anaerobic  jar.  After  10  days  at  33.5°  C.  the  spirochaetes  had 
multiplied  considerably,  in  all  but  one  case,  together  with  bacteria.  He 
obtained  pure  cultures  from  these  initial  cultivations  after  much  diffi- 
culty, by  a  number  of  methods.  At  first  he  succeeded  only  by  allowing 
the  spirochaetes  to  grow  through  Berkefeld  filters,  which  they  did  on 
the  fifth  day.  A  better  method  more  recently  adopted  by  him  consists 
in  preparing  higl:i  tubes  of  three  parts  of  very  slightly  alkaline  or  neutral 
agar  to  which  a  piece  of  sterile  tissue  has  been  added.  These  tubes  are 
then  inoculated  from  the  impure  cultures  with  a  long  pipette.  Close 
to  the  tissue  and  along  the  stab  the  spirochaetes  and  bacteria  will  grow 
and,  after  about  ten  days  to  two  weeks,  the  spirochaetes  will  have  wan- 
dered away  from  the  stab  and  will  be  visible  as  hazy  colonies.    They  can 

^Noguchi,  Jour.  Exp.  Med.,  xiv,  1911;  xvii,  1913. 


DISEASES  CAUSED  BY  SPIROCH.ETES  601 

then  be  fished,  after  cutting  the  tubes,  and  directly  transplanted  to 
other  serum-agar-tissue  tubes  prepared  as  before,  and  eventually  will 
grow  in  pure  culture.  By  this  method  Noguchi  has  also  cultivated  pure 
cultures  from  lesions  in  monkeys,  and  has  produced  lesions  both  in  rab- 
bits and  monkeys  with  his  pure  cultures.  He  has  thus  for  the  first  time 
carried  out  Koch's  postulates  with  syphilis  and  established  beyond  the 
shadow  of  a  doubt  the  etiological  significance  of  Spirochseta  pallida  in 
syphilis. 

The  writer,  with  Hopkins,  has  successfully  applied  Noguchi's  method 
and  has  found  that,  after  once  cultivated  artificially,  the  treponema 
pallidum  can  be  obtained  in  quantity  best  by  cultivation  in  flasks  con- 


FiG.  131. — SpiROCHiETA  PALLIDA.     Livcr,  congenital  syphilis. 
(Levaditi  method.) 

taining  heated  or  unheated  rabbit  kidney  with  ascitic  broth  and  sealed 
with  paraffin.  Recently  we  have  been  using  modifications  of  a  method 
worked  out  in  our  laboratory  by  Miss  Gilbert,  in  which  slanted  egg, 
with  or  without  glycerin,  made  as  for  tubercle  cultivation,  is  used  in- 
stead of  kidney  tissue.  This  is  put  up  in  high  tubes  and  ascitic  broth 
and  paraffin  oil  added.  By  this  method,  large  quantities  of  culture, 
pallida  are  obtained  within  two  weeks  and  can  be  concentrated  in  large 
quantities. 

Animal  Pathogenicity. — Until  very  recently,  all  experimental  inoc- 
ulation of  animals  was  unsuccessful.  During  the  year  1903  Metchnikoff 
and  Roux  ^  finally  succeeded  in  transmitting  the  disease  to  monkeys. 
The  monkey  first  used  by  these  observers  was  a  female  chimpanzee. 
At  the  point  of  inoculation,  the  clitoris,  there  appeared,  twenty-six  days 
after  inoculation,  a  typical  indurated  chancre,  which  was  soon  followed 
by  swelling  of  the  inguinal  glands.  Fifty-six  days  after  the  inoculation 
there  appeared  a  typical  secondary  eruption,  together  with  swelling  of 
the  spleen  and  of  the  lymph  nodes.    Similar  successful  experiments  were 

1  Metchnikoff  et  Roux,  Ann.  de  Tinst.  Pasteur,  1903,  1904,  and  1905. 


602  PATHOGENIC  MICROORGANISMS 

made  soon  after  this  by  Lassar.^  Soon  after  the  experiments  of  Metch- 
nikoff  and  Roux,  successful  inoculations  upon  lower  monkeys  (maca- 
cus)  were  carried  out  by  Nicolle.^  Since  that  time,  it  has  been  found 
by  various  observers  that  almost  all  species  of  monkeys  are  susceptible. 
Simple  subcutaneous  injection  is  not  sufficient  to  produce  a  lesion. 
The  technique  which  has  given  the  most  satisfactory  results  consists 
in  the  cutaneous  implantation  of  small  quantities  of  syphilitic  tissue 
obtained  by  excision  or  curetting  of  primary  and  secondary  lesions.  A 
small  pocket  is  made  under  the  mucous  membrane  of  the  genitals  or  of 
the  eyebrows  and  the  tissue  placed  in  this  under  aseptic  precautions. 
The  inoculation  may  be  made  directly  from  the  human  being,  but  can 
also  be  successfully  carried  out  from  monkey  to  monkey  for  many 
generations.  Attempts  at  transmission  from  tertiary  lesions  have  so  far 
been  unsuccessful.  The  spirochsetes  can  be  demonstrated  both  in  the 
primary  lesions  of  the  inoculated  animal  and  in  the  secondarily  enlarged 
glands.  The  successful  inoculation  of  rabbits  with  syphilis  has  been 
recently  performed  by  Bertarelli.^  He  obtained  ulcerative  lesions  by 
inoculation  upon  the  cornea  and  into  the  anterior  chamber  of  the  eye 
and  was  able  to  prove  the  syphilitic  nature  of  these  lesions  by  finding 
the  spirochsete  within  the  tissue.  In  these  animals,  as  well  as  in  the 
lower  monkeys,  the  disease  usually  remains  localized. 

In  1907,  Parodi  showed  that  syphilitic  lesions  could  be  produced  by 
direct  inoculation  into  the  testicles  of  rabbits.  This  method  of  inocula- 
tion has  been  subsequently  studied  by  many  investigators,  especially 
by  Uhlenhuth  and  Mulzer.^  It  is  the  easiest  method  of  obtaining 
spirochaete  in  any  quantity  from  lesions  in  man.  The  spirochaete-con- 
taining  lesions  may  be  either  excised  or  scraped  as  conditions  permit 
and  rubbed  up  in  a  mortar  with  sterile  sand,  in  a  few  centimeters  of 
sterile  human  ascitic  fluid.  This  emulsion  is  then  injected  directly 
into  the  substance  of  rabbit  testicles.  A  swelling  supervenes  which 
is  often  noticeable  after  two  weeks,  and  is  usually  at  its  height  in  5  to 
7  weeks.  At  this  time  the  testicle  is  much  larger  than  normal,  some- 
times evenly  swollen  and  sometimes  nodular,  and  of  a  firm  elastic  con- 
sistency. When  taken  out  at  castration  it  oozes  a  sticky  fluid,  both 
from  testicle  and  tunica,  which  is  rich  in  actively  motile  spirochaetes. 
By.  continuous  transinoculation  from  one  rabbit  to  another  such  a  strain 
can  be  indefinitely  carried  along.     It  can  be  inoculated  from  rabbits 

1  Lassar,  Berl.  klin.  Woch.,  xl,  1903.  ^  Nicolle,  Ann.  de  I'inst.  Pasteur,  1903. 

» Bertarelli,  Cent.  f.  Bakt.,  xli,  1906. 

^  Uhlenhuth  und  Mvlzer,  Arb.  a.  d.  k.  Gesimdh'tsamt.,  xxxiii,  1909. 


DISEASES  CAUSED  BY  SPIROCHiETES  603 

to  monkeys  and  vice  versa.  This  method  as  well  as  Noguchi's  cul- 
tivations have  opened  a  new  era  of  spirochsete  investigation.  It  is 
stated  by  some  observers  that  intravenous  inoculation  of  rabbits  may 
be  followed  by  localization  in  the  testis  and  occasionally  gummatous 
infections  in  other  parts  of  the  body  have  been  induced  after  such  in- 
oculation by  Uhlenhuth,  Mulzer,  and  others. 

Immunization  in  Syphilis. — It  is  a  well-known  fact  observed  by 
clinicians  that  during  active  syphilis  the  patient  cannot  be  superin- 
fected.  That  this  resistance  develops  quite  rapidly  was  shown  by 
Metchnikoff  and  Roux,  who  found  that  reinfection  of  a  monkey  was 
possible  if  attempted  within  two  weeks  of  the  first  inoculation,  but 
was  unsuccessful  if  delayed  beyond  this  period. 

On  the  basis  of  this  knowledge,  Metchnikoff,^  Finger  and  Land- 
steiner,^  and  others  have  made  attempts  to  devise  some  method  of  im- 
munization. They  attempted  to  attenuate  the  syphilitic  virus  by  re- 
peated passage  through  monkeys.  These  experiments  were  unsuccess- 
ful, the  last-mentioned  observers  finding  absolutely  no  attenuation 
after  twelve  generations  of  monkey  inoculation. 

Bertarelli  and  others  have  shown  that  the  production  of  a  syphilitic 
lesion  on  the  cornea  of  one  eye  does  not  protect  against  an  inoculation 
done  on  the  other.  Rabbits  that  haye  been  inoculated  jvith  spirochsete 
material  and  that  have  not  developed  syphilitic  disease  can  be  success- 
fully inoculated  on  subsequent  attempts.  The  offspring  of  female  rab- 
bits with  syphilis  of  the  cornea  are,  according  to  Muhlens,  not  immune. 

There  is  no  evidence  so  far  that  specific  therapy  or  treatment  with 
spirochaete  material   has  had  favorable   influence  upon  the  disease. 
Chemotherapy   has    had    results    analogous    to   those    obtained    in- 
man.^ 

Attempts  at  passive  immunization  have  been  entirely  without  success. 

Investigations  carried  on  in  our  own  laboratory  in  the  last  three 
years  have  shown  definitely,  we  think,  that  immunization  of  animals 
with  culture  pallida  produces  antibodies,  agglutinins,  treponemacidal 
substances,  entirely  analogous  to  similar  substances  produced  against 
bacteria.  However,  there  is  a  biological  change  which  takes  place 
when  treponema  pallidum  is  cultivated.  The  antibodies  produced 
with  the  culture  pallida  have  no  action  whatsoever  upon  the  virulent 

1  Metchnikoff,  Arch.  g^n.  de  m6d.,  1905. 

2  Finger  und  Landsteiner,  Sitzungsber.  d.  Wien.  Akad.  d.  Wiss.,  1905. 

» Von  Prowazek,  "Handbuch  der  pathogenen  Protozoen,"  i,  1912,  Leipzig,  Bartscli. 


604  PATHOGENIC  MICROORGANISMS 

organisms.  The  latter,  indeed,  seem  to  be  entirely  insulated  against 
such  antibodies  and  do  not  induce  antibody  formation  to  any  great 
extent,  in  either  the  infected  animal  or  man.  Both  active  and  passive 
immunization  with  culture  pallida  and  the  sera  produced  with  them 
have  no  effect.  We  have  obtained  some  evidence,  however,  that  in  rab- 
bits a  purely  local  resistance  developes  in  the  tissue  praviously  the  site 
of  a  lesion. 

The  occurnence  of  a  Wassermann  reaction  was  formerly  supposed  to 
indicate  the  existence  of  specific  syphilitic  antibodies  in  the  serum  of 
patients.  More  recent-  information  regarding  this  reaction  seems  to 
show  that  it  depends  upon  the  presence  in  the  serum  of  syphilitic 
patients  of  substances  produced  indirectly  because  of  the  presence  of 
syphilitic  infection.  It  may  be  a  relative  increase  of  globulins  or,  as 
Schmidt  has  suggested,  a  change  in  the  physical  state  of  the  globulins 
or  other  substances  present  in  the  serum.  At  any  rate  it  has  been  found 
that  the  fixation  of  complement  in  the  Wassermann  reaction  does  not 
depend  upon  the  occurrence  of  a  specific  antigen-antibody  reaction. 
In  the  first  place  the  antigens  most  commonly  used,  and  successfully 
so,  in  the  Wassermann  reactions,  are  non-specific  lipoidal  extracts  of 
normal  organs. 

Again  it  has  been  demonstrated  that  extracts  of  cultures  of  the 
Spirochseta  pallida  as  well  as  extractions  from  the  testes  of  syphilitic 
rabbits  do  not  furnish  an  antigen  suitable  for  the  Wassermann  reaction. 
This  has  followed  especially  from  the  work  of  Noguchi,^  of  Craig  and 
Nichols,^  and  ourselves.  This  forms  a  corollary  to  the  other  experi- 
ments previously  mentioned  and  shows  that,  whatever  the  Wassermann 
reaction  may  be,  it  is  not  a  specific  complement  fixation  in  the  sense  of 
Bordet  and  Gengou.  It  must  be  admitted,  therefore,  that  our  knowl- 
edge of  syphilis  immunity  is  in  its  infancy  and  that  we  know  very  little 
about  the  systemic  reactions  which  follow  infection  with  the  Spirochseta 
pallida. 

The  fact  that  the  syphilitic  virus  does  not  pass  through  a  filter  has 
been  demonstrated  by  Klingmiiller  and  Baermann,^  who  inoculated 
themselves  with  filtrates  from  syphilitic  material. 


^Zinsser  and  Hopkins,  Jour,  of  Exp.  Med.,  xxi,  1915,  p.  576;  xxiii,  1916,  p.  323; 
xxiii,  1916,  p.  329;  xxiii,  1916,  p.  341. 

2  Noguchi,  Jour.  Am.  Med.  Assoc,  1912. 

3  Craig  and  Nichols,  Jour.  Exp.  Med.,  xvi,  1912. 

*  Klingmiiller  und  Baermann,  Deut.  med.  Woch.,  1904. 


DISEASES  CAUSED  BY  SPIROCH.ETES 


605 


THE  SPIROCHiETES  OF  RELAPSING  FEVER 

The  microorganisms  causing  relapsing  'fever  were  first  observed  in 
1873,  by  Obermeier/  who  demonstrated  them  in  the  blood  of  patients 
suffering  from  this  distinct  type  of  fever.     Since  his  time  extensive 


^0 


Fig.  132. — Spirochete  of  Relapsing  Fever, 

and  Flournoy.) 


(After  Norris,  Pappenheimer, 


studies  by  many  other  observers  have  proven  beyond  question  the 
etiological  connection  between  the  disease  and  the  organisms. 

Morphology  and  Staining. — The  spirochsete  of  Obermeier  is  a  delicate 
spiral  thread  measuring  from  7  to  9  mi  era  in  length  (Novy),  and  about 
1  micron  in  thickness.  While  this  is  its  average  size,  it  may,  according 
to  some  observers,  be  considerably  longer  than  this,  its  undulations 
varying  from  4  to  10  or  more  in  number.     Compared  with  the  red  blood 

^Obermeier,  Cent.  f.  d.  med.  Wiss.,  11,  1873. 


606 


PATHOGENIC  MICROORGANISMS 


cells  among  which  they  are  seen,  the  microorganisms  may  vary  from 
one -half  to  9  or  10  times  the  diameter  of  a  corpuscle.  In  fresh  prepara- 
tions of  the  blood,  very  active  corkscrew-hke  motility  and  definite  lateral 
oscillation  are  observed.  In  stained  preparations  no  definite  cellular 
structure  can  be  made  out,  the  cell  body  appearing  homogeneous,  except 
in  degenerated  individuals,  in  which  irregular  granulation  or  beading 
has  been  observed.    Flagella  have  been  described  by  various  observers. 


Fig.  133. — Spiroch.ete  of  Relapsing  Fever.      Citrated  normal  rat  blood. 
(After  Norris,  Pappenheimer,  and    Flouriioy.) 


Novy  and  Knapp^  believe  that  the  organisms  possess  only  one  terminal 
flagellum.  Zettnow,^  on  the  other  hand,  claims  to  have  demonstrated 
lateral  flagella  by  special  methods  of  staining.  Norris,  Pappenheimer, 
and  Flournoy,^  in  smears  stained  by  polychrome  methods,  have  described 
long,  filamentous  tapering  ends  which  they  interpreted  as  bipolar, 
terminal  flagella,  never  observing  more  than  one  at  each  end.  Spores 
are  not  found. 

Cultivation. — Innumerable  attempts  to  induce  these  microorganisms 
to  multiply  upon  artificial  media  have  been  made.  Novy  and  Knapp 
succeeded  in  keeping  the  microorganisms  alive  and  virulent  in  the 

^  Novy  and  Knapp,  Jour,  of  Infec.  Dis.,  3,  1906. 

2  Zettnow,  Deut.  med.  Woch.,  32,  1906. 

3  Norris,  Pappenheimer,  and  Flournoy,  Jour,  of  Inf.  Dis.,  3,  1906. 


DISEASES  CAUSED  BY  SPIROCILETES 


607 


original  blood  for  as  long  as  forty  days,  and  call  attention  to  the  fact 
that  the  length  of  time  for  which  they  may  be  kept  aUve  depends  to  a 
great  extent  upon  the  stage  of  fever  at  which  the  blood  is  removed  from 
the  patient.  They  do  not,  however,  believe  that  extensive  multiphca- 
tion,  or,  in  other  words,  actual  cultivation,  had  taken  place  in  their 
experiments.  Norris,  Pappenheimer,  and  Flournoy,  on  the  other  hand, 
have  obtained  positive  evidence  of  multiplication  of  the  spirochaetes  in 
fluid  media.  They  obtained  their  cultures  by  inoculating  a  few  drops 
of  spirochaetal  rat  blood  into  3  to  5  c.c.  of  citrated  human  or  rat  blood. 
Smears  made  from  these  tubes,  after  preservation  for  twenty-four  hours 
at  room  temperature,  showed  the  microorganisms  in  greater  number 


Fig.  134. — SpiROCHiETE  of  Relapsing  Fever.     (From  preparation  furnished 
by  Dr.  G.  N.  Calkins.) 


than  in  the  original  infected  blood.  A  similar  multiplication  could  be 
observed  in  transfers  made  from  these  '^ first-generation"  tubes  to  other 
tubes  of  citrated  blood.  Attempts  at  cultivation  for  a  third  generation, 
however,  failed. 

Noguchi  ^  has  lately  successfully  cultivated  the  spirochaete  of  Ober- 
meier  in  ascitic  fluid  containing  a  piece  of  sterile  rabbit's  kidney  and  a 
few  drops  of  citrated  blood  under  anaerobic  conditions. 

Four  different,  probably  distinct  varieties  of  spirochsete  have  been 
described  in  connection  with  relapsing  fever,  all  of  which  have  been 
cultivated  by  Noguchi  by  means  of  this  method.  The  first  is  known  as 
the  spirochsete   of    Obermeier  mentioned  above.      Probably  distinct 


40 


^Noguchi,  Jour.  Exp.  Med.,  xvii,  1913. 


608  PATHOGENIC  MICROORGANISMS 

are  the  Spirochseta  Duttoni,  described  by  Dutton  and  Todd^  inl905,  the 
Spirochseta  Kochi,  and  the  Spirochaeta.Novyi,^  the  organism  studied  by 
Norris  and  Flournoy  and  Pappenheimer,  and  regarded  as  a  different 
species  by  them. 

Pathogenicity. — Inoculation  with  blood  containing  these  spirochsetes 
produces  disease  in  monkeys,  rats,  and  mice.  Attempts  to  transmit 
the  disease  experimentally  to  dogs,  rabbits,  and  guinea-pigs  have  so 
far  been  unsuccessful.  The  subcutaneous  inoculation  of  monkeys  is 
followed  after  from  two  to  four  days  by  a  rise  of  temperature  which 
occurs  abruptly  as  is  the  case  in  the  disease  in  man  and  which  may  last 
several  days.  During  this  time  the  spirochsetes  can  be  found  in  the 
blood  of  the  animals  just  as  it  is  found  in  that  of  infected  human  beings. 
The  temperature  subsides  after  a  day  or  more,  when  it  again  rapidly 
returns  to  normal.  As  a  rule,  the  paroxysms  are  not  repeated.  Occa- 
sionally, however,  two  or  three  attacks  may  supervene  before  immunity 
is  established.  In  rats,  an  incubation  time  of  from  two  to  five  days 
occurs.  At  the  end  of  this  time  the  spirochsetes  may  be  found  in  large 
numbers  in  the  blood,  and  the  animals  show  symptoms  of  a  severe 
systemic  infection.  The  attack  lasts  from  four  to  five  days,  at  the  end 
of  which  time  the  microorganisms  again  disappear.  Occasionally  even 
in  these  animals  relapses  have  been  observed.  Gross  pathological 
changes  are  not  found,  with  the  exception  of  an  enlargement  of  the 
spleen. 

In  man  the  disease  caused  by  the  spirochsete  of  Obermeier,  commonly 
known  as  relapsing  fever,  is  common  in  India,  Africa,  and  most  of  the 
warmer  countries.  It  has,  from  time  to  time,  been  observed  epidemically 
in  Europe,  especially  in  Russia,  and  a  few  epidemics  have  occurred  in 
the  Southern  United  States.  The  disease  comes  on  abruptly,  beginning 
usually  with  a  chill  accompanied  by  a  sharp  rise  of  temperature  and  gen- 
eralized pains.  Together  with  the  rise  of  temperature,  which  often  ex- 
ceeds 104°  F.,  there  are  great  prostration  and  occasioifally  delirium.  Early 
in  the  disease  the  spleen  becomes  palpable  and  jaundice  may  appear. 
The  spirochsetes  are  easily  detected  in  the  blood  during  the  persistence 
of  the  fever,  which  lasts  usually  from  three  to  ten  days.  At  the  end 
of  this  time  the  temperature  usually  drops  as  suddenly  as  it  rose,  and 
the  general  symptoms  rapidly  disappear.  After  a  free  interval  of 
from  one  to  three  weeks  a  relapse  may  occur,  which  is  usually  less 
severe  and  of  shorter  duration  than  the  original  attack.     Two,  three,  or 

1  Dutton  and  Todd,  Brit.  Med.  Jour.,  1905. 
^  Novy  and  Fraenkel,  cited  from  Noguchi. 


DISEASES  CAUSED  BY  SPIROCHiETES 


609 


even  four  attacks  may  occur,  but  the  disease  is  not  very  often  fatal. 
When  patients  do  succumb,  however,  the  autopsy  findings  are  not 
particularly  characteristic.  Apart  from  the  marked  enlargement  of 
the  spleen,  which  histologically  shows  the  changes  indicatin"g  simple 
hyperplasia,  and  a  slight  enlargement  of  the  Hver,  no  lesions  are  found. 
The  diagnosis  is  easily  made  during  the  febrile  stage  by  examination  of 
a  small  quantity  of  blood  under  a  cover-slip  or  in  the  hanging-drop 
preparation. 

Several  types  of  relapsing  fever  have  been  described.  In  Africa  the 
disease  has  long  been  prevalent  in  many  regions  and  the  investigations 
of  Ross  ami  Milne, ^  Koch,^  Button  and  Todd,^  and  others  have  brought 


Fig.  135. — Spiroch^ete  of  Dutton,  African  Tick  Fever.     (From  prepara- 
tion furnished  by  Dr.  G.  N.  Calkins.) 

to  light  that  many  conditions  occurring  among  the  natives,  formerly 
regarded  as  malarial,  are  caused  by  a  species  of  spirochsete.  Whether" 
or  not  the  microorganisms  observed  m  the  African  disease  are  exactly 
identical  with  the  spirochaete  observed  by  Obermeier  is  yet  a  question 
about  which  several  opinions  are  held.  Dutton  and  Todd  believe  that 
the  same  microorganism  is  responsible  for  both  diseases.  Koch,  on  the 
other  hand,  beUeves  that  the  slightly  smaller  size  of  the  African  spiro- 
chaete and  the  milder  course  of  the  clinical  symptoms  indicate  a  defi- 
nite difference  between  the  two.  Animal  experiments  made  with  the 
African  organism,  furthermore,  usually  show  a  much  more  severe  in- 
fection than  do  similar  inoculations  with  the  European  variety.     The 


1  Ross  and  Milne,  Brit.  Med.  Jour.,  1904. 

2  Koch,  Deut.  med.  Woch.,  xxxi,  1905. 

3  DiUton  and  Todd,  Lancet,  1905,  and  Jour,  of  Trop.  Med.,  1906. 


610  PATHOGENIC  MICROORGANISMS 

spirochsete  found  in  the  African  disease  is  usually  spoken  of  at  present 
as  "Spirochaeta  Duttoni.''  Novy  and  Knapp/  after  extensive  studies 
with  the  microorganisms  from  various  sources,  have  come  to  the  conclu- 
sion that,'  although  closely  related,  definite  species  differences  exist  be- 
tween the  two  types  mentioned  above,  and  that  these  again  are  definitely 
distinguished  from  similar  organisms  described  by  TurnbulP  as  occurring 
in  a  similar  disease  observed  in  India. 

The  mode  of  transmission  of  this  disease  is  not  clear  for  all  types. 
Dutton  and  Todd,  however,  were  able  to  show  satisfactorily  that,  in  the 
case  of  the  African  disease  at  least,  transmission  occurs  through  the 
intermediation  of  a  species  of  tick.  The  conditions  under  which  such 
intermediation  occurs  have  been  carefully  studied  by  Koch.^  The 
tick  (Ornithodorus  moubata)  infects  itself  when  sucking  blood  from 
an  infected  human  being.  The  spirochsete  may  remain  alive  and 
demonstrable  within  the  body  of  the  tick  for  as  long  as  three  days. 
Koch  has  shown,  furthernore,  that  they  may  be  found  also  within  the 
eggs  laid  by  an  infected  female  tick.  He  succeeded  in  producing  experi- 
mental infection  in  monkeys  by  subjecting  the  animals  to  the  bites 
of  the  infected  insects.  For  the  European  variety  of  the  disease  no 
such  intermediate  host  has  as  yet  been  demonstrated. 

Immunity. — It  has  long  been  a  well-known  fact  that  recovery  from 
an  attack  of  relapsing  fever  usually  results  in  a  more  or  less  definite 
immunity.  The  blood  of  human  beings,  monkeys,  and  rats  which  have 
recovered  from  an  attack  of  this  disease  show  definite  and  specific 
bactericidal  and  agglutinating  substances,  and  Novy  and  Knapp  have 
demonstrated  that  the  blood  serum  of  such  animals  may  be  used  to 
ponfer  passive  immunity  upon  others. 


VINCENT'S  ANGINA 

The  condition  known  as  Vincent^s  angina  consists  of  an  inflamma- 
tory lesion  in  the  mouth,  pharynx,  or  throat,  situated  most  frequently 
upon  the  tonsils.  The  disease  usually  begins  as  an  acute  stomatitis, 
pharyngitis,  or  tonsillitis,  which  soon  leads  to  the  formation  of  a  pseudo- 
membrane,  which,  at  this  stage,  has  a  great  deal  of  resemblance  to  that 
caused  by  the  diphtheria  bacillus.  At  later  stages  of  the  disease  there 
may  be  distinct  ulceration,  the  ulcers  having  a  well-defined  margin 


1  Novy  and  Knapp,  loc.  cit.  2  Tumbull,  Indian  Med.  Gaz.,  1905. 

» Koch,  Berl.  med.  Woch.,  1906. 


DISEASES  CAUSED  BY  SPIROCH.ETES 


611 


and  "punched-out"  appearance,  so  that  clinically  they  have  of  ten  been 
erroneously  diagnosed  as  syphilis.  Apart  from  the  localized  pain,  the 
disease  is  usually  mild,  but  occasionally  moderate  fever  and  systemic 
disturbances  have  been  observed.  Unlike  diphtheria  and  syphilis,  this 
peculiar  form  of  angina  usually  yields,  without  difficulty,  to  local  treat- 
ment. 

The  nature  of  lesions  of  this  peculiar  kind  was  not  clear  until  Plaut,^ 


i 


.    ^^   •  V.        \       x,/       *|  '       .    *    *l| 


¥  i 


^1    . 


Fig.  136. — Smear  prom  the  throat  of  a  Case  of  Vincent's  Angina. 

Giemsa  Stain. 

Vincent,^  and  others  reported  uniform  bacteriological  findings  in  cases 
of  this  description.  These  observers  have  been  able  to  demonstrate 
in  smears  from  the  lesions  a  spindle-shaped  or  fusiform  bacillus,  to- 
gether with  which  there  is  usually  found  a  spirillum  not  unlike  the 
spirillum  of  relapsing  fever.      The   two  microorganisms    are    almost 

1  Plant,  Deut.  med.  Woch.,  xlix,  1894. 

2  Vincent,  Ann.  de  I'inst.  Pasteur,  1896,  and  Bull,  et  m6m.  de  la  see.  med.  des 
h6p.  de  P.,  1898. 


612*  PATHOGENIC  MICROORGANISMS 

always  found  together  in  this  form  of  disease  and  were  regarded  by 
the  first  observers  as  representing  two  distinct  forms  dwelHng  in  sym- 
biosis. More  recently  Tunnicliff/  on  the  basis  of  experimental  work, 
has  claimed  identity  for  the  two  forms,  believing  that  they  represent 
different  developmental  stages  of  the  same  organism. 

The  fusiform  hacilli  described  by  Vincent,  Plant,  Babes,  and  others, 
are  from  3  to  10  micra  in  length,  and  have  a  thickness  at  the  center 
varying  from  0.5  to  0.8  micron.     From  the  center  they  taper  gradually 


^ 


./ 


) 

r 

^ 

\ 


Fig.  137. — ^Throat  Smear.  Vincent's  Angina.    Fusiform  bacilli  and  spirilla. 

toward  the  ends,  ending  in  blunt  or  sharp  points.  The  length  of  these 
bacilli  may  vary  greatly  within  one  and  the  same  smear  preparation. 
They  are  usually  straight,  sometimes  slightly  curved.  They  do  not  stain 
very  easily  with  the  weaker  anilin  dyes,  but  are  readily  stained  by 
Loeffler's  methylene-blue,  carbol-fuchsin,  or  better,  by  Giemsa's  stain. 
Stained  by  Gram,  they  are  usually  decolorized,  though  in  this  respect  the 
writers  have  found  them  to  vary.  Stained  preparations  show  a  charac- 
teristic inequality  in  the  intensity  of  the  stain,  the  bacilli  being  more 

1  Tunnidiff,  Jour,  of  Infec.  Dis.,  3,  1906.  . 


DISEASES  CAUSED  BY  SPIROCH.ETES  613 

deeply  stained  near  the  end,  and  showing  a  banded  or  striped  alternation 
of  stained  and  unstained  areas  in  the  central  body.  Their  staining 
quaUties  in  this  respect  are  not  unlike  those  of  the  diphtheria  bacillus, 
and  according  to  Babes  ^  the  dark  areas  are  to  be  interpreted  as  meta- 
chromatic granules.     The  bacilli  are  not  motile. 

The  spirilla  found  in  Vincent's  angina  are  usually  somewhat  longer 
than  the  fusiform  bacilli,  and  are  made  up  of  a  variable  number  of  un- 
dulations, shallow  and  irregular  in  their  curvatures,  unlike  the  more 
regularly  steep  waves  of  Spirochseta  pallida.  They  are  stained  with 
even  more  difficulty  than  are  the  bacilli  and  usually  appear  less  distinct 
in  the  preparations.  The  stain,  however,  is  taken  without  irregu- 
larity, showing  none  of  the  apparent  metachromatism  observed  in  the 
bacilli. 

By  the  earlier  observers  cultivation  of  these  microorganisms  was 
attempted  without  success.  Recently,  however,  it  has  been  shown  that 
cultivation  could  be  carried  out  under  anaerobic  conditions.  Tunni- 
cliff  ^  has  cultivated  the  organisms  anaerobically  upon  slants  of  ascitic 
agar  at  37.5°  C.  This  observer  found  that  in  such  cultures,  before  the 
fifth  day,  bacilli  only  could  be  found,  that  after  this  time,  however, 
spirilla  gradually  appeared  and  finally  constituted  the  majority  of  the 
organisms  in  the  culture.  It  appeared  to  TunnicHff  from  this  study 
that  the  spirilla  might  be  developed  out  of  the  fusiform  microorgan- 
isms representing  the  adult  form. 

The  microorganisms  of  Vincent's  angina,  when  occurring  in  the 
throat,  are  rarely  present  alone,  being  usually  accompanied  by  other 
microorganisms,  such  as  staphylococci,  streptococci,  and  not  infre- 
quently diphtheria  bacilli.  When  occurring  together  with  diphtheria, 
they  are  said,  by  some  German  observers,  to  aggravate  the  latter 
condition  considerably.  This  frequent  association  with  other  micro- 
organisms renders  it  impossible  to  decide  conclusively  that  the  fusi- 
form bacilli  and  spirilla  are  the  primary  etiological  factors  in  these 
inflammations.  It  has  been  frequently  suggested  that  they  may  be 
present  as  secondary  invaders  upon  the  soil  prepared  for  them  by  other 
microorganisms. 

Animal  inoculation  with  these  microorganisms  has  led  to  little  result. 

Fusiform  Bacilli  other  than  those  in  Vincent's  Angina. — ^Fusiform 
bacilli  morphologically  indistinguishable  from  those  found  in  the  angina 
of  Vincent  may  frequently  be  found  in  smears  taken  from  the  gums, 

*  Babes,  in  KoUe  und  Wassermann,  1 .  Erganzungsband,  1907. 
2  TunnicHff,  Jour,  of  Infec.  Dis.,  3,  1906. 


614  PATHOGENIC  MICROORGANISMS 

from  carious  teeth,  and  occasionally  among  the  microorganisms  in  the 
pus  from  old  sinuses.  Several  varieties  of  these  bacilli  have  been  de- 
scribed in  connection  with  definite  pathological  conditions. 

Babes/  in  1893,  observed  spindle-shaped  bacilli  not  unlike  those 
described  above,  but  somewhat  shorter,  in  histological  sections  prepared 
from  tissues  from  the  gums  of  individuals  suffering  from  scurvy.  He 
found  similar  bacilli  in  rabbits  intravenously  inoculated  with  material 
from  the  patients  and  was  able  to  cultivate  the  bacilli  for  several  genera- 
tions. His  descriptions,  however,  of  the  microorganisms  as  found  in  the 
secondary  cultures  vary  considerably  from  those  of  the  original  findings 
in  the  gums  of  the  patients.     His  results  are  not  convincing. 

In  noma,  a  gangrenous  disease  of  the  gums  and  cheeks,  occurring 
occasionally  in  individuals  who  have  been  severely  run  down  by  acute 
infectious  diseases  or  great  hardship.  Weaver  and  Tunnicliff  have  found 
spirilla  and  fusiform  bacilli  in  large  numbers.  The  organisms  were  pres- 
ent not  only  in  smears  from  the  surface,  but  were  also  found  by  histo- 
logical methods,  in  large  numbers,  lying  in  the  tissues  beyond  the 
area  of  necrosis.  Here  again  it  is  not  entirely  certain  whether  these 
microorganisms  were  the  primary  etiological  factors  or  whether 
they  are  to  be  regarded  merely  as  secondary  invaders  of  a  necrotic 
focus. 

Fusiform  bacilli  are  cultivated  with  greater  ease  than  formerly  sup- 
posed; we  have  found  it  relatively  simple  to  grow  them  together  with 
Gram  positive  cocci  in  symbiosis  in  simple  broth  tubes  covered  with 
paraffin  oil  without  the  addition  of  any  enriching  substance  and  in 
similar  symbiotic  conditions  on  infusion  agar  plates  under  incomplete 
anaerobic  conditions.  In  such  plates  they  form  curious  colonies  in 
which  the  fusiform  bacilli  and  micrococci  are  intimately  commingled. 
Krumwiede  ^  has  had  no  difficulty  in  cultivating  them  in  pure  culture 
in  anaerobic  plates. 

SPIROCHUETA  PERTENUIS 

In  a  disease  known  as  ^'Framboesia  tropica,"  or  popularly  "Yaws," 
occurring  in  tropical  and  subtropical  countries  and  much  resembling 
syphilis,  Castellani,^  in  1905,  was  able  to  demonstrate  a  species  of 
spirochsete  which  has  a  close  morphological  resemblance  to  Spirochseta 
pallida.     The  microorganism  was  found  in  a  large  percentage  of  the  cases 

1  Babes,  Deut.  med.  Woch.,  xliii,  1893. 

^Krumwiede,  Jour.  Inf.  Dis.,  1913. 

3  CasteUani,  Brit.  Med.  Jour.,  1905,  and  Deut.  med.  Woch.,  1906. 


DISEASES  CAUSED  BY  SPIROCHETES  615 

examined  both  in  the  cutaneous  papules  and  in  ulcerations.  Confirm- 
atory investigations  on  a  larger  series  of  cases  were  later  carried  out  by 
von  dem  Borne. ^ 

The  microorganism  is  from  7  to  20  micra  in  length  with  numerous 
undulations  and  pointed  ends.  Examined  in  fresh  preparations,  it  has 
an  active  motility  similar  to  that  of  Spirochaeta  paUida.  In  smears 
it  is  easily  stained  by  means  of  the  Giemsa  method. 

Both  the  clinical  similarity  between  yaws  and  syphilis,  as  well  as 
the  similarity  between  the  microorganisms  causing  the  diseases,  has 
opened  the  question  as  to  the  identity  of  the  two  microorganisms. 
According  to  most  clinical  observers,  however,  yaws,  which  is  a  disease 
characterized  chiefly  by  a  generalized  papular  eruption,  is  unquestion- 
ably distinct,  clinically,  from  lues,  and  experiments  of  Neisser,  Baermann, 
and  Halberstadter,2as  well  as  of  Castellani  ^  himself,  have  tended  to  show 
that  there  is  a  distinct  difference  between  the  immunity  produced  by 
attacks  of  the  two  diseases.  The  disease  is  transmissible  to  monkeys, 
as  is  syphihs,  but  it  has  been  satisfactorily  shown  that  monkeys  inocu- 
lated with  syphilitic  material,  while  no  longer  susceptible  to  infection 
with  Spirochaeta  pallida,  may  still  be  successfully  inoculated  with 
Spirochaeta  pertenuis.  I 

SPIROCHAETA  GALLINARUM 

An  acute  infectious  disease  occurring  among  chickens,  chiefly  in 
South  America,  has  been  shown  by  Marchoux  and  Salimbeni^  to  be 
caused  by  a  spirochaete  which  has  much  morphological  similarity  to  the 
spirochaete  of  Obermeier. 

The  disease  comes  on  rather  suddenly  with  fever,  diarrhea,  and  great 
exhaustion,  and  often  ends  fatally.  The  spirochaete  is  easily  demon- 
strated in  the  circulating  blood  of  the  animals  by  staining  blood-smears 
with  Giemsa's  stain  or  with  dilute  carbol-fuchsin. 

Artificial  cultivation  of  the  microorganism  has  not  yet  been  ac- 
complished. Experimental  transmission  from  animal  to  animal  is  easily 
carried  out  by  the  subcutaneous  injection  of  blood.  Other  birds,  such 
as  geese,  ducks,  and  pigeons,  are  susceptible;  mammals  have,  so  far, 
not  been  successfully  inoculated.     According  to  the  investigations  of 

1  Von  dem  Borne,  Jour.  Trop.  Med.,  10,  1907. 

2  Neisser,  Baermann,  und  Halherstadter,  Miinch.  med.  Woch.,  xxviii,  1906. 

3  Castellani,  Jour,  of  Hyg.,  7,  1907. 

*  Marchovx  et  Salimbeni,  Ann.  de  Tinst.  Pasteur,  1903. 


616 


PATHOGENIC   MICROORGANISMS 


Levaditi  and  Manouelian/  the  spirochsetes  are  found  not  only  in  the 
blood  but  thickly  distributed  throughout  the  various  organs. 

Under  natural  conditions,  infection  of  chickens  seems  to  depend 
upon  a  species  of  tick  which  acts  as  an  intermediate  host  and  causes 
infection  by  its  bite.     The  spirochsete,  according  to  Marchoux  and  Sal- 

imbeni,  may  be  found  in  the  intes- 
tinal canal  of  the  ticks  for  as  long 
as  five  months  after  their  infection 
from  a  diseased  fowl. 

In  the  blood  of  animals  which 
have  survived  an  infection,  agglutin- 
ating substances  appear  and  active 
immunization  of  animals  may  be 
carried  out  by  the  injection  of  in- 
fected blood  in  which  the  spirochsetes 
have  been  killed,  either  by  moderate 
heat  or  by  preservation  at  room 
temperature.  The  serum  of  immune 
animals,  furthermore,  has  a  pro- 
tective action  upon  other  birds. 

It  is  not  impossible  that  the  Spiro- 
chseta  gallinarum  may  be  identical 
with  the  Spirochseta  anserina  previ- 
ously discovered  by  Sacharoff  .^  This 
last-named  microorganism  causes  a  disease  in  geese,  observed  espe- 
cially in  Russia  and  Northern  Africa,  which  both  clinically  and  in  its 
pathological  lesions  corresponds  closely  to  the  disease  above  described 
as  occurring  in  chickens.  The  spirochsete  is  found  during  the  febrile 
period  of  the  disease  in  the  circulating  blood,  is  morphologically  indis- 
tinguishable from  the  spirochsete  of  chickens,  and  can  not  be  cultivated 
artificially.  The  similarity  is  further  strengthened  by  the  fact  that 
Spirochaeta  anserina  is  pathogenic  for  other  birds,  but  not  for  animals 
of  other  genera.  Noguchi  has  succeeded  in  cultivating  Spirochaeta 
gallinarum  by  the  same  method  by  which  he  has  cultivated  the  or- 
ganisms of  relapsing  fever.  Ascitic  fluid  tubes  with  a  piece  of  sterile 
rabbit  kidney  were  inoculated  with  a  few  drops  of  blood  containing  the 
spirochsetes  and  cultivated  at  37.5°  C.  under  anaerobic  conditions. 
Spirochaeta  phagedenis. — This  is  an  organism  cultivated  by  Noguchi 

1  Levaditi  et  Manouelian,  Ann.  de  I'inst.  Pasteur,  1906. 

2  Sacharoff,  Ann.  de  I'inst.  Pasteur,  1891. 


Fig.  138. — Spiroch^ta  gallina- 
rum .  (From  preparation  furnished 
by  Dr.  G.  N.  Calkins.) 


DISEASES   CAUSED   BY    SPIROCHiETES  617 

by  his  ascitic-fluid-tissue  method  from  phagedenic  lesions  on  human 
external  genitals.     It  is  probably  a  new  species. 

Spirochseta  macrodentium. — Cultivated  by  Noguchi;  *  is  believed  by 
him  to  be  identical  with  the  spirochsete  found  in  Vincent's  angina. 

Spirochseta  microdentium. — ^A  similar  organism  with  wide  con- 
volutions, cultivated  by  Noguchi  from  the  tooth  deposits  chiefly  in 
children.  It  was  grown  on  mixtures  of  sheep  serimi  water  and  sterile 
tissue  in  a  way  similar  to  that  employed  by  him  for  other  organisms  of 
this  group. 

SpirochsBta  calligyrum. — Cultivated  by  Noguchi  ^  from  condylomata 
— ^is  probably  a  new  species. 

Weil's  Disease. — Weil's  disease  is  a  malady  which  has  been  known 
for  a  long  time,  in  which  there  is  a  moderate  febrile  movement,  with 
jaundice,  enlarged  liver,  and  a  hemorrhagic  eruption.  In  this  disease, 
Inada,  Yutaka,  Hoki,  Kaneko,  and  Ito  ^  have  described  the  Spiro- 
chaete  icterohaemoragica.  They  have  found  the  organisms  in  the  liver 
by  the  Levaditi  method,  the  adrenal  glands  and  the  kidneys,  and  have 
transmitted  it,  with  the  blood  of  cases,  to  guinea-pigs  by  intraperi- 
toneal injection.  They  succeeded  in  cultivating  it  by  the  Noguchi 
method. 

Rat-Bite  Fever. — Rat-bite  fever  is  a  peculiar  disease,  which,  after  an 
incubation  period  of  ten  or  more  days,  is  characterized  by  fever,  head- 
ache and  inflammation  at  the  site  of  the  bite,  swollen  lymph  glands, 
skin  eruption  and  pains.  After  three  to  six  days  the  fever  ceases  and 
an  afebrile  period  of  two  or  three  days  ensues.  After  this  the  fever 
again  occurs.  Recently  Futaki,  Takaki,  Taniguchi,  and  Osumi  *  have 
described  a  treponema  which  they  have  called  Treponema  morsus  muris: 
It  is  a  spiral  organism,  somewhat  larger  than  the  Treponema  pallidum, 
and  is  found  in  the  skin,  the  lymph  nodes,  and  in  the  blood.  They 
have  succeeded  in  inoculating  rats  and  have  cultivated  it  in  Schi- 
mamine  medium,  which  consists  of  100  c.c.  of  horse  serum  in  which 
0.5  to  0.75  gram  of  sodium  nucleate  is  dissolved  and  carbon  dioxide 
passed  through  the  solution  until  the  serrmi  becomes  transparent.  It 
is  then  heated  for  three  days  at  60°,  and  on  the  fourth  day  at  65° 
until  it  coagulates.  This  medium  is  deeply  inoculated,  but  no  other 
anaerobic  precautions  are  taken. 

*  Noguchi,  Jour.  Exp.  Med.,  xv,  1912.        *  Noguchi,  Jour.  Exp.  Med.,  xvii,  1913. 
'  Jour,  of  Exp.  Med.,  1916,  xxiii,  p,  249.    *  Jour.  Exp.  Med,,  1916,  xxiii,  p,  377. 


H;  GLENN  BfilL 


CHAPTER  XLIV 
THE  HIGHER  BACTERIA 
{Chlamydobacteriacece,  Trichomycetes) 

Standing  midway  between  the  true  bacteria  and  the  more  complex 
molds  or  Hyphomycetes,  there  are  a  number  of  pathogenic  micro- 
organisms which  offer  great  difficulties  to  classification.  In  the  classifi- 
cation of  Migula  most  of  these  forms  have  been  placed  in  a  rather 
heterogeneous  group,  the  Chlamydobacteriacese.  By  other  authors, 
notably  Lachner-Sandoval,^  Berestnew,^  and  by  Petruschky,^  the  close 
relationship  of  these  forms  to  the  higher  hyphomycetes  has  been  em- 
phasized and  they  have  been  grouped  as  a  subdivision  of  the  true  molds 
under  the  family  name  of  Trichomycetes. 

Petruschky''  proposes  the  following  clear  schematization,  which, 
even  though  possibly  defective  from  a  purely  botanical  point  of  view, 
is  at  least  serviceable  for  the  purposes  of  the  bacteriologist. 

Hyphomycetes 


True  molds  Trichomycetes 


Leptothrix  Cladothrix  Streptothrix  Actinomyces 

Leptothrix  is  used  to  designate  those  forms  which  appear  as  simple 
threads  without  branching. 

Cladothrix  is  a  thread-like  form  in  which  false  branching  may  be 
recognized.  By  false  branching  is  meant  an  appearance  resulting  from 
the  fragmentation  of  threads.  The  terminal  cell  breaks  away  from  the 
main  stem,  is  set  at  an  angle  by  the  elongation  of  the  thread  itself,  and, 

^  Lachner-Sandoval,  "Ueber  Strahlenpilze."     Diss.  Strassburg,  1898. 

2  Berestnew,  Ref.  Cent.  f.  Bakt.,  xxiv,  1898. 

3  Petnischky,  in  KoUe  imd  Wassermann,  "Handbuch,"  etc. 
*  Petruschky,  loc.  cit. 

618 


THE  HIGHER  BACTERiA*  619 

as  both  continue  dividing,  the  simulation  of  true  branching  is  pro- 
duced. 

Streptothrix  denotes  forms  with  numerous  true  branches  and  spores 
which  usually  appear  in  chains. 

Actinomyces  is  of  more  complicated  structure,  characterized  by  the 
formation  of  club-shaped  ends  and  the  stellate  arrangement  of  its 
threads. 

LEPTOTHRIX 

Members  of  the  leptothrix  group  have  been  observed  in  connection 
with  inflammations  of  the  mouth  and  pharynx  by  Frankel,^  Michelson,^ 
Epstein,^  and  others.  In  many  of  these  cases  the  organism  was  identi- 
fied by  morphology  chiefly,  pure  cultures  not  having  been  obtained. 
The  disease  in  none  of  these  cases  was  accompanied  by  severe  systemic 
symptoms  and  it  is  likely  that  when  found  in  human  beings  the  organ- 
isms may  be  regarded  simply  as  comparatively  harmless  saprophytes 
appearing  in  connection  with  some  other  specific  inflammation. 

Cultivation  of  the  Leptothrices  is  not  easy  and  has  been  successful 
only  in  the  hands  of  Vignal  *  and  Arustamoff .^ 

GLADOTHRIX 

Owing  to  much  confusion  in  the  differentiation  of  these  forms  from 
the  streptothrices,  it  is  not  possible  to  determine  whether  cases  of  true 
cladothrix  infection  have  been  observed.  It  is  likely  that  most  cases 
ascribed  to  microorganisms  of  this  class  have  really  been  due  to  strep- 
tothrix infection.  The  deciding  criterion  is,  of  course,  the  formation  of 
branches  and  these  seem  to  have  been  observed  in  most  of  the  cases 
described.  A  closer  differentiation,  in  the  future,  between  true  and 
false  branching  can  alone  determine  whether  or  not  cases  of  cladothrix 
infection  proper  may  occur. 

STREPTOTHRIX 

Reports  of  cases  of  streptothrix  infection  of  various  parts  of  the 
body,  in  both  animals  and  man,  are  abundant  in  the  literature.     The 

*  Frdnkel,  Eulenburg's  "  Realencycl.  d.  gesam.  Heilkunde,"  1882. 
2  Michelson,  Berl.  klin.  Woch.,  ix,  1889. 

»  Epstein,  Prag.  med.  Woch.,  1900. 

•  Vignal,  Ann.  de  phys.,  viii,  1886. 

»  Arustamoff,  Quoted  from  Petruschky,  loc.  cit 


620 


PATHOGENIC  MICROORGANISMS 


earliest  observations  were  made  upon  microorganisms  isolated  from  the 
human  conjunctiva.  Nocard  *  in  1888  described  a  member  of  this 
group  as  the  etiological  factor  in  a  disease  "  farcies  du  boeuf "  occurring 
among  cattle  in  Guadeloupe.  Eppinger  ^  found  streptothrices  in  the  pus 
of  a  cerebral  abscess.     Petruschky,^  Berestneff,'*  Flexner,^  Norris  and 


Fig    139. — Cladothrix,  Showing  Fausb  Bbanching. 


Larkin,®  and  a  number  of  other  observers  have  found  these  microor- 
ganisms in  cases  of  pulmonary  disease,  simulating  tuberculosis.  Sup- 
purations of  bone  and  of  the  skin  and  the  intestinal  canal  have  been 
reported.  The  infection,  therefore,  is  not  very  rare,  but  the  diverse 
experiences  of  workers  who  have  attempted  to  cultivate  these  micro* 

»  Nocard,  Ann.  de  Finst.  Pasteur,  ii,  1888. 

a  Eppinger,  Wien.  klin.  Woch.,  1890. 

s  Petruschky,  Verhandl.  d.  Kongr.  f.  innere  Mediz.,  1898. 

*Berestneff,  Zeit.  f.  Hyg.,  xxix,  1898. 

*Flexner,  Jour.  Exp.  Med.,  iii,  1896. 

•  Norris  and  Larkin,  Proc.  of  N.  Y.  Path.  Soc,  March,  1899. 


THE  HIGHER  BACTERIA 


621 


organisms  seem  to  indicate  that  not  all  of  the  incitants  described  be- 
longed to  one  and  the  same  variety,  but  that  probably  a  number  of 
different  types  may  exist. 

Morphology. — Morphologically  the  streptothrices  show  considerable 
variation.  In  material  from  infectious  lesions  they  have  most  often 
appeared  as  rods  and  filaments  with  well-marked  branching.  Occasion- 
ally the  filaments  are  long  and  interwined,  and  branches  have  shown 
bulbous  or  club-shaped  ends.  In  Norris  and  Larkin^s  case,  the  young 
cultures  in  the  first  generations  seem  to  have  consisted  chiefly  of  rod- 
shaped  forms  not  uYi'.'ke  bacilh  of  the  diphtheria  group,  showing  marked 
metachromatisn:  when  stained  with  Loeffler's  methylene-blue.    They  are 


Fig.  140. — Streptothrix,  Showing  True  Branching. 


easily  stained  with  this  dye  or  with  aqueous  fuchsin.     In  tissue  sections 
they  may  be  demonstrated  by  the  Gram-Weigert  method. 

Cultivation.— Direct  cultivation  upon  agar  and  gelatin  plates  has 
occasionally  been  successful.  At  the  end  of  four  or  five  days  grayish- 
white,  glistening,  flat  colonies  may  appear  which  attain  a  diameter  of 
several  millimeters  within  two  weeks.  The  colonic  later  may  take  on  a 
yellowish  hue  and  begin  to  liquefy  the  gelatin.  In  bouillon  flocculent 
precipitates  and  surface  pellicles  of  interwined  threads  may  form,  with- 
out clouding  of  the  medium.  Norris  and  Larkin*  found  much  difficulty 
in  cultivating,  but  finally  succeeded  by  making  smears  of  the  infectious 
material   upon   fresh,    sterile   kidney-tissue   of   rabbits.     The   micro- 

»  Norris  and  Larkin,  loc.  dt. 


622  PATHOGENIC  MICROORGANISMS 

organisms  grew  abundantly  upon  this,  but  failed  to  grow  on  any  of  the 
other  tissues.  After  growth  of  several  generations  upon  this  medium, 
cultures  were  finally  obtained  upon  agar  plates  and  upon  broth. 

Inoculation  of  cultures  into  rabbits  and  guinea-pigs  have  given  rise 
to  subcutaneous  abscesses,  bronchopneumonia,  and  suppuration,  accord- 
ing to  the  mode  of  infection. 


ACTINOMYCES 

Among  the  diseases  caused  by  the  Trichomycetes  or  higher  bacteria, 
the  most  important  is  actinomycosis.  Occurring  chiefly  in  some  of  the 
domestic  animals,  notably  in  cattle,  the  disease  is  observed  in  man  with 
sufficient  frequency  to  make  it  of  great  clinical  importance.  In  cattle 
the  specific  microorganism  which  gives  ries  to  the  disease  was  first 
observed  by  Bollinger*  in  1877.  In  the  following  year  Israel^  dis- 
covered a  similar  microorganism  in  human  cases. 

The  parasites  appear  in  the  pus  from  discharging  lesions  as  small 
granular  bodies,  plainly  visible  to  the  naked  eye  and  somewhat  resem- 
bling sulphur  granules,  of  a  grayish  or  of  a  pale  yellow  color.  In  size 
they  measure  usually  a  fraction  of  a  millimeter.  Ordinarily  they  are 
soft  and  easily  crushed  under  a  cover-slip,  but  occasionally,  especially  in 
old  lesions,  they  may  be  quite  hard,  owing  to  calcification. 

Microscopically  they  are  most  easily  recognized  in  fresh  preparations 
prepared  by  crushing  the  granules  upon  the  slide  under  a  cover-slip  and 
examining  them  without  staining.  They  may  be  rendered  more  clearly 
visible  by  the  addition  of  a  drop  or  two  of  20  per  cent  potassium  hydrate. 
When  the  granules  are  calcareous,  the  addition  of  a  drop  of  concentrated 
acetic  acid  will  facilitate  examination.  Fresh  preparations  may  be 
examined  after  staining  with  Gram's  stain.  Observed  under  the  micro- 
scope, the  granules  appear  as  rosette-like  masses,  the  centers  of  which 
are  quite  opaque  and  dense,  appearing  to  be  made  up  of  a  closely  meshed 
network  of  filaments.  Around  the  margins  there  are  found  radially 
arranged  striations  which  in  many  cases  end  in  characteristically  club- 
shaped  bodies.  Inside  of  the  central  network  there  are  often  seen 
coccoid  or  spore-like  bodies  which  have  been  variously  interpreted  as 
spores,  as  degeneration  products,  and  as  separate  cocci  fortuitously 
found  in  symbiosis  with   the  actinomyces.     Individually  considered, 

»  Bollinger,  Deutsch.  Zeit.  f.  Thiermed.,  iii,  1877. 
2  Israel  Virch.  Arch.,,  74, 1878,  and  78, 1879. 


THE  HIGHER  BACTERIA  623 

the  central  filaments  have  approximately  the  thickness  of  an  anthrax 
bacillus  and  are,  according  to  Babes/  composed  of  a  sheath  within 
which  the  protoplasm  contains  numerous  and  different  sized  granules. 

About  the  periphery  of  the  granules  the  free  ends  of  the  filaments 
become  gradually  thickened  to  form  the  so-called  actinomycosis  "  clubs." 
These  clubs,  according  to  most  observers,  must  be  regarded  as  hyaline 
thickenings  of  the  sheaths  of  the  threads  and  are  believed  to  represent  a 
form  of  degeneration  and  not,  as  some  of  the  earlier  observers  believed, 
organs  of  reproduction.  They  are  homogeneous,  and  in  the  smaller  and 
presumably  younger  granules  are  extremely  fragile  and  soluble  in  water. 
In  older  lesions,  especially  in  those  of  cattle,  the  clubs  are  more  re- 
sistant and  less  easily  destroyed. 

They  appear  only  in  the  parasites  taken  from  active  lesions  in  animals 
or  man,  or,  as  Wright  ^  has  found,  from  cultures  to  which  animal  serum 
or  whole  blood  has  been  added.  In  cultures  from  media  to  which  no 
animal  fluids  have  been  added,  such  as  glucose  agar  or  gelatin,  no  clubs 
are  found.  In  preparations  stained  by  Gram's  method  the  clubs  give 
up  the  gentian-violet  and  take  counter-stains,  such  as  eosin. 

The  coccus-like  bodies  found  occasionally  lying  between  the  filaments 
of  the  central  mass,  most  observers  now  agree,  do  not  represent  any- 
thing comparable  to  the  spores  of  the  true  hyphomycetes.  In  many 
cases  they  are  unquestionably  contaminating  cocci;  in  others  again 
they  may  represent  the  results  of  degeneration  of  the  threads. 

In  tissue  sections,  the  microorganisms  'may  be  demonstrated  by 
Gram's  method  of  staining  or  by  a  special  method  devised  by  Mallory.^ 
This  is  as  follows  for  paraffin  sections : 

1.  Stain  in  saturated  aqueous  eosin  10  minutes. 

2.  Wash  in  water. 

3.  Anilin  gentian- violet,  5  minutes. 

4.  Wash  with  normal  salt  solution. 

5.  Gram's  iodin  solution  1  minute. 

6.  Wash  in  water  and  blot. 

7.  Cover  with  anilin  oil  until  section  is  clear. 

8.  Xylol,  several  changes. 

9.  Mount  in  balsam. 

Cultivation. — The  isolation  of  actinomyces  from  lesions  may  be 
easy  or  difficult  according  to  whether  the  pus  is  free  from  contamination 

»  Babes,  Virch.  Arch.,  105,  1886. 
2  J.  H.  Wright,  Jour.  Med.  Res.,  viii,  1905. 

»  Mallory,  Method  No.  1,  Mallory  and  Wright,  "  Path.  Technique,"  Phila.,  1908. 
41 


624 


PATHOGENIC  MICROORGANISMS 


1 


or  whether  it  contains  large  numbers  of  other  bacteria.  In  the  latter 
case  it  may  be  almost  impossible  to  obtain  cultures.  The  descriptions 
of  methods  of  isolation  and  of  cultural  characteristics  given  by  various 
writers  have  shown  considerable  differences.  The  most  extensive 
cultural  work  has  been  done  by  Bostroem/  Wolff  and  Israel,  and  by  J. 
H.  Wright.  Bostroem  has  described  his  cultures  as  aerobic,  but  Wolff 
and  Israel  ^  and  Wright  ^  agree  in  finding  that  the  microorganisms  iso- 


FiG.   141. — ^Actinomyces  Granule    Crushed   Beneath  a   Cover-glass.     Un- 
stained.   Low  power.    Shows  radial  striations.     (After  Wright  and  Brown.) 

lated  by  them  from  actinomycotic  lesions  grow  but  sparsely  under  aerobic 
conditions  and  favor  an  environment  which  is  entirely  free  from  oxygen, 
or  at  least  contains  it  only  in  small  quantities.  The  method  for  isolation 
recommended  by  Wright  is,  briefly,  as  follows:  Pus  is  obtained,  if 
possible,  from  a  closed  lesion  and  washed  in  sterile  water  or  broth.  The 
granules  are  then  crushed  between  two  sterile  slides  and  examined  for 

1  Bostroem,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.,  ix,  1890. 

2  Wolff  und  Israel,  Virch.  Arch.,  126,  1891. 
»/.  H.  Wright,  Jour.  Med.  Res.,  viii,  1905. 


THE  HIGHER  BACTERIA 


625 


the  presence  of  filaments.  If  these  are  present  in  reasonable  abundance, 
the  material  is  distributed  in  tubes  of  glucose  agar,  which  are  then 
allowed  to  solidify.  If  these  first  cultivations  show  a  large  number  of 
contaminations,  Wright  recommends  the  preservation  of  other  washed 
granules  in  test  tubes  for  several  weeks,  in  the  hope  that  contaminating 
microorganisms  may  thus  be  killed  by  drying 
before  the  actinomyces  lose  their  viability. 

If  cultivation  is  successful  colonies  will  ap- 
pear, after  two  to  four  days  at  37.5°  C,  as  minute 
white  specks,  which,  in  Wright^s  cultures,  ap- 
peared most  abundantly  within  a  zone  situated 
5  to  10  millimeters  below  the  surface  of  the 
medium.  Above  and  below  this  zone  they  are 
less  numerous,  indicating  that  a  small  amount  of 
oxygen  furnishes  the  best  cultural  environment. 
Upon  the  surface  of  agar  slants,  growth,  if  it 
takes  place  at  all,  is  not  luxuriant. 

In  alkaline  meat-infusion  broth  growth 
takes  place  in  the  form  of  heavy,  flocculent 
masses  which  appear  at  the  bottom  of  the  tubes. 
Surface  growth  and  clouding  do  not  take  place. 

Milk  and  potato  have  been  used  as  culture 
media  but  are  not  particularly  favorable. 

Pathogenicity. — As  stated  above,  actinomy- 
cosis occurs  spontaneously  most  frequently 
among  cattle  and  human  beings.  It  may  also 
occur  in  sheep,  dogs,  cats,  and  horses.  Its  loca- 
tions of  predilection  are  the  various  parts 
adjacent  to  the  mouth  and  pharynx.  It  occurs 
also,  however,  in  the  lungs,  in  the  intestinal 
canal,  and  upon  the  skin.  When  occurring  in 
its  most  frequent  location,  the  lower  jaw,  the 
disease  presents,  at  first,  a  hard  nodular  swell- 
ing which  later  becomes  soft  because  of  central  necrosis.  It  often 
involves  the  bone,  causing  a  rarefying  osteitis.  As  the  sweUings 
break  down,  sinuses  are  formed  from  which  the  granular  pus  is 
discharged.  The  neighboring  lymph  nodes  show  painless,  hard  swell- 
ings. Histologically,  about  the  filamentous  knobs  or  granules,  there  is  a 
formation  of  epithelioid  cells  and  a  small  round-cell  infiltration.  In 
older  cases  there  may  be  an  encapsulation  in  connective  tissue  and  a 


Fig.  142. — Actino- 
myces Granule 
Crushed  Beneath  a 
Cover-glass.  .U  n  - ' 
stained.  The  prepara- 
tion shows  the  margin 
of  the  granule  and  the 
"clubs."  (After Wright 
and  Brown.) 


626 


PATHOGENIC  klCROORGANISMS 


calcification  of  the  necrotic  masses,  leading  to  spontaneous  cure.  As  a 
rule,  this  process  is  extremely  chronic.  Infection  in  the  lungs  or  in  the 
intra-abdominal  organs  is,  of  course,  far  more  serious.  When  death 
occurs  acutely,  it  is  often  due  to  secondary  infection.  The  disease  is 
acquired  probably  by  the  agency  of  hay,  straw,  and  grain.  Berestnew  ^ 
has  succeeded  in  isolating  actinomyces  from  straw  and  hay  which  he 
covered  with  sterile  water  in  a  potato  jar  and  placed  in  the  incubator. 
After  a  few  days  small  white  specks  looking  like  chalk  powder  appeared 
upon  the  stalks,  which,  upon  further  cultivation,  he  was  able  to  identify 
as  the  organism  in  question. 

Animal  inoculation,  carried  out  extensively  both  with  pus  and  with 
pure  cultures  by  several  observers,  has  yielded  little  result.     Progressive 


Fig.  143. — ^Branching  Filaments  of  Actinomyces.    (After  Wright  and  Brown.) 

actinomycotic  lesions  were  never  obtained,  although  occasionally  small 
knolls  containing  colonies  surrounded  by  epithelioid  cells  and  connective 
tissue  were  observed,  showing  that  the  invading  microorganisms  were 
able  to  survive  and  grow  for  a  short  time,  but  were  not  sufficiently 
virulent  to  give  rise  to  an  extensive  disease  process.  Transmission  from 
animal  to  animal,  or  from  animal  to  man  directly,  has  not  been  satis- 
factorily proven. 

Whether  or  not  there  are  various  forms  of  actinomyces  must 
as  yet  be  regarded  as  an  open  question.  The  investigations  of 
Wolff  and  Israel,  however,  together  with  those  of  Wright,  who  alone 
observed  thirteen  different  strains,  seem  to  indicate  that  most,  if 
not  all,  of  the  cases  clinically  observed  are  due  to  one  and  the  same 
microorganism. 


»  Berestnew,  Ref.  Cent.  f.  Bakt.,  24,  1898. 


THE  HIGHER  BACTERIA  627 


MYCETOMA  (MADURA   FOOT) 

The  disease  known  by  this  name  is  not  unhke  actinomycosis.  It 
is  more  or  less  strictly  limited  to  warmer  climates  and  was  first  recog- 
nized as  a  clinical  entity,  in  India,  by  Carter.^  Clinically  it  consists 
of  a  chronic  productive  inflammation  most  frequently  attacking  the 
foot,  less  often  the  hand,  very  infrequently  other  parts  of  the  body. 
Nodular  swellings  occur,  which  break  down  in  their  centers,  leading  to 
the  formation  of  abscesses,  later  of  sinuses.  Often  the  bones  are  in- 
volved and  a  progressive  rarefying  osteitis  results.  From  the  sinuses  a 
purulent  fluid  exudes,  in  which  are  found  characteristic  granular  bodies. 
These  may  be  hard,  brittle,  and  black,  resembling  grains  of  gunpowder, 
or  may  be  grayish-white  or  yellow  and  soft  and  grumous.  According  to 
the  appearance  of  these  granules,  two  varieties  of  the  disease  are  dis- 
tinguished, the  "melanoid"  and  the  "ochroid.^'  Many  observers 
believe  that  the  yellow  or  ochroid  variety  is,  in  fact,  actinomycosis. 
The  black  variety,  which  is  certainly  a  distinct  disease,  is  caused  by  a 
member  of  the  hyphomycetes  group.  The  parasite  has  been  carefully 
studied  by  Wright,^  from  whose  description  the  following  points  are 
taken : 

The  small,  brittle  granules  observed  under  the  microscope  show  a 
dark,  almost  opaque  center  along  the  edges  of  which,  filaments,  or 
hyphse,  may  be  seen  in  a  thickly  matted  mass.  By  crushing  the  granules 
Ainder  a  cover-slip  in  a  drop  of  sodium  hypochlorite  or  of  strong  sodium 
hydrate,  the  black  amorphous  pigment  is  dissolved  and  the  structural 
elements  of  the  fungus  may  be  observed.  They  seem  to  be  composed 
of  a  dense  meshwork  of  mycelial  threads  which  are  thick  and  often 
swollen,  and  show  many  branches.  Transverse  partitions  are  placed 
at  short  distances  and  the  individual  filaments  may  be  very  long. 
Spores  were  not  observed  by  Wright.  In  a  series  of  over  fifty  cultiva- 
tions on  artificial  media  from  the  original  lesion,  Wright  obtained  growth 
in  a  large  percentage. 

In  broth,  he  obtained  at  first  a  rapid  growth  of  long  hyphse  which 
eventually  formed  a  structure  which  he  compares  in  appearance  to  a 
powder-puff. 

On  agar,  growth  appeared  within  less  than  a  week  and  spread  over 
the  surface  of  the  medium  as  a  thick  meshwork  of  spreading  hyphae 

1  Carter  on  My<cetoma,  etc.,  London,  1874. 
«  Wright,  Jour,  of  Exper.  Med.,  3,  1898. 


628  PATHOGENIC  MICROORGANISMS 

of  a  grayish  color.  In  old  cultures  black  granules  appeared  among 
the  mycelial  meshes. 

On  'potato,  he  observed  a  dense  velvety  membrane,  centrally  of  a  pale 
brown,  white  at  the  periphery.  Small  brown  droplets  appeared  on  the 
growth  in  old  cultures. 

Animal  inoculation  with  this  microorganism  has  so  far  been  un- 
successful. 


CHAPTER  XLV 
THE  YEASTS 
{Blastomycetes,  Saccharomycetes) 

The  yeasts  or  blastomycetes  form  a  distinctive  family  among  uni- 
cellular microorganisms,  characterized  essentially  by  their  method  of 
multiplication  by  budding.  By  this,  they  are  sharply  separated  from 
the  bacteria.  Their  differentiation  from  the  higher  fungi,  the  hypho- 
mycetes,  however,  is  less  definitely  established,  since  the  chief  character- 
istic of  this  latter  class,  the  formation  of  hyphae  and  mycelial  threads,  has 
occasionally  been  described  for  some  of  the  forms  otherwise  identified 
with  the  yeasts.  It  is  probable  that  a  gradual  transition  between  the 
two  families  exists,  represented  by  a  number  of  connecting  forms,  some- 
times spoken  of  as  oidia.  For  the  practical  purposes  of  the  bacteriolo- 
gist, the  yeast  family  is  sufficiently  distinct,  both  morphologically  and 
biologically,  to  make  a  separate  classification  extremely  useful. 

The  yeast  cell,  as  a  rule,  is  oval,  but.  among  the  wild  yeasts,  or 
'  torulae,"  spherical  forms  are  common.  In  size,  great  variations  occur, 
but  in  general  the  yeasts  are  much  larger  than  bacteria,  measuring  usually 
from  10  to  20  micra  in  length  with  a  width  of  about  one-half  or  two-thirds 
of  the  long  diameter.  They  possess  a  weU-defined,  doubly-contoured" 
cell-membrane,  composed  chiefly  of  cellulose,  and  their  body  protoplasm, 
unlike  tha,t  of  the  bacteria,  shows  definite  structure.  Within  a  mass 
of  finely  granul*ar cytoplasm,  a  number  of  highly  refractive  globules  and 
vacuoles  may  be  observed.  Some  of  the  globules  have  been  interpreted 
as  fat-droplets.  Other  granules,  revealed  by  special  staining  methods, 
are  interpreted  as  nuclear  material. 

When  budding  takes  place,  the  mother  cell  sends  out  a  small, 
globular  evagination  of  the  cell  membrane  into  which  maternal  proto- 
plasm flows.  This  bud  gradually  enlarges  until  it  has  attained  approxi- 
mately the  same  size  as  the  original  cell.  Until  that  time,  free  inter- 
communic'ation  between  the  protoplasm  of  mother  and  daughter  cell 
exists,  l^inally ,  by  gradual  narrowing  of  the  isthmus  connecting  the 
two^'th^"  ii^ughter  cell  becomes  complete  and  free.     By  some  observers 

629 


630  PATHOGENIC  MICROORGANISMS 

• 

definite  karyokinetic  changes  in  the  nuclear  structures  have  been  de- 
scribed as  accompanying  the  budding  process.  This  observation,  how- 
ever, has  not  been  generally  confirmed.  Under  conditions  of  special 
stress,  such  as  unfavorable  environment  or  lack  of  nutrition,  most 
yeasts  possess  the  power  of  forming  spores.  These,  called  "  ascospores,'' 
are  formed  within  the  yeast  cell  itself,  each  spore  forming  a  separate 
membrane  of  its  own,  but  all  of  them  lying  well  protected  within  the 
original  cell-membrane.  The  number  of  ascospores  formed  is  constant 
for  each  species,  and  rarely  exceeds  four. 

The  yeasts  have  been  studied  most  extensively  in  connection  with 
fermentation  and  are  industrially  of  great  importance  in  the  production 


Fig.  144. — ^Yeast  Cells.    Young  culture  unstained.    (After  Zettnow.) 

of  wine  and  beer.  Although  Schwann,  as  early  as  1837,  recognized  the 
fact  that  many  fermentations  could  not  occur  without  the  presence  of 
yeast,  it  was  not  until  considerably  later  that  the  study  of  yeast  fermen- 
tation was  put  upon  a  scientific  basis.  The  typical  fermentative  action 
consists  in  the  transformation  of  sugar  into  ethyl  alcohol  according  to 
the  following  formula: 

Ce  H,2  Oe  =  2  C^  Hg  OH  +  2  CO^ 

The  enzyme  by  which  this  fermentation  is  produced  is  known  as  "zy- 
mase," and  is,  according  to  Buchner,  in  most  cases,  an  endo-enzyme 
which  may  be  procured  from  the  cell  by  expression  in  a  hydraulic 
press.     In  addition  to  this,  however,  the  various  yeasts  also  produce 


THE  YEASTS  631 

other  ferments  by  means  of  which  they  may  split  higher  carbohydrates, 
such  as  saccharose,  maltose,  and  even  starch,  and  prepare  them  for 
action  of  the  zymase.  The  manner  in  which  this  is  accomplished,  and 
the  by-products  which  are  formed  during  the  process,  vary  among 
different  species,  and  it  is  for  this  reason  that  the  employment  of  pure 
cultures  is  of  such  great  importance  in  the  wine  and  beer  industries 
where  differences  in  flavor  and  other  qualities  may  be  directly  dependent 
upon  the  particular  species  of  yeast  employed  for  the  fermentation.  It 
is  due  to  the  work  chiefly  of  Pasteur  ^  and  of  Hansen  ^  that  the  beer  and 
wine  industries  have  been  carried  on  along  exact  and  scientific  lines. 

As  the  incitants  of  disease  in  man,  the  yeasts  have  been  much  studied 
since  1894,  when  Busse  ^  reported  a  case  of  fatal,  generalized  yeast  in- 
fection, beginning  from  a  tibial  bone  abscess.  The  microorganism  which 
was  found  in  this  case  he  named  "  Saccharomyces  hominis."  In  morpho- 
logical and  biological  characters  it  appeared  to  be  a  typical  yeast,  grew 
readily  upon  most  artificial  media,  and  produced  active  fermentation  in 
sugars.  Mycelia  were  not  observed.  When  inoculated  into  animals, 
this  yeast  proved  pathogenic  for  mice  and  rats.  A  peculiarity  of 
Busse's  culture,  observed  since  then  in  the  case  of  many  pathogenic 
yeasts,  was  the  formation  of  gelatinous  capsules,  of  varying  thicknesses, 
about  the  individual  cells,  developing  with  particular  luxuriance  in  the 
animal  lesions. 

In  1896,  Gilchrist  "*  described  a  peculiar  skin  disease,  which  he  spoke 
of  as  pseudo-lupus  vulgaris,  in  the  lesions  of  which  he  demonstrated  a 
large  number  of  round,  doubly-contoured  bodies  which,  though  differing 
somewhat  from  Busse's  saccharomyces,  were  unquestionably  members 
of,  or  closely  related  to,  the  family  of  blastomycetes. 

In  the  same  year,  Curtis,^  in  France,  isolated  a  similar  form  from  a 
myxoma  of  the  leg.  Ophiils^  has  described  a  number  of  fatal  cases 
occurring  in  California,  which  at  first  were  wrongly  interpreted  as 
protozoan  in  origin,  but  later  were  determined  by  him  to  be  caused 
by  a  species  of  blastomycetes.  In  a  case  observed  by  Zinsser  ^  simi- 
lar microorganisms  were  isolated  from  an  abscess  of  the  back,  which 

»  Pasteur,  "  ^fitudes  sur  la  bi^re,"  Paris,  1876. 

2  Hansen,  "  Prac.  Studies  in  Fermentation,"  London,  1896. 

3  Busse,  Cent.  f.  Bakt.,  I,  xvi,  1894,  and  Virch.  Arch.,  140,  1895. 
*  Gilchrist,  Bull.  Johns  Hopkins  Hosp.,  vii,  1896. 

8  Curtis,  Ann.  de  I'inst.  Pasteur,  10,  1896. 

« Ophiils,  Jour.  Exp.  Med.,  6,  1901.     , 

7  Zinsser,  Proc.  N.  Y.  Path.  Soc,  vii.  1907, 


632  PATHOGENIC  MICROORGANISMS 

in  almost  all  respects  corresponded  to  Gilchrist's  cultures.  Animal 
inoculation  in  rabbits  and  guinea-pigs  proved  positive  in  this  case  and 
the  organism  seemed  to  show  selective  action  for  the  lungs  and  spleen. 
In  the  lungs  of  the  animals,  especially,  lesions  were  found  with  surpris- 
ing regularity  even  when  the  inoculation  was  made  intraperitoneally. 

Cases  of  blastomycotic  infection  in  man  have  been  reported  in  large 
numbers  and  appear  to  be  less  rare  than  they  were  formerly  believed  to 
be.  The  clinical  course  of  the  disease  is  by  no  means  uniform.  A  well- 
defined  clinical  picture  seems  to  characterize  the  cases  of  blastomycotic 
dermatitis  first  described  by  Gilchrist.  The  eruption  is  very  chronic 
and  begins  usually  as  a  small  pimple  or  papule  with  moderate  induration 
of  the  skin.  Scabs  and  pustules  then  form,  which  discharge  yellowish- 
white  pus.  As  the  lesion  slowly  spreads,  the  older  areas  show  a  tendency 
to  spontaneous  healing.  In  Gilchrist's  ^  case,  it  took  four  years  for  the 
lesion  to  spread  two  inches.  When  not  purely  cutaneous,  blastomycotic 
infection  takes  the  form  of  chronic  abscess  formation  occurring  in  various 
parts  of  the  body.  In  the  latter,  metastatic  lesions  in  the  lungs  have 
been  occasionally  observed,  and  in  one  case  cited  by  Ophiils,^  the  lung 
seemed  to  have  been  the  primary  focus. 

The  fact  that  blastomycetes  have  frequently  been  found  in  tumor 
tissue  has  led  several  Italian  observers^  to  assume  an  etiological 
relationship  between  these  microorganisms  and  malignant  growths. 
Absolutely  no  satisfactory  evidence  in  favor  of  such  a  belief  has 
been  advanced,  however,  and  the  yeasts  in  these  conditions  must  be 
regarded  as  purely  fortuitous  findings. 

In  animals,  diseases  caused  by  members  of  the  yeast  family  have 
been  reported  by  various  observers.  The  most  important  communica- 
tion of  this  kind  is  by  Tokishige,*  who  found  these  microorganisms  in  a 
nodular  skin  disease  occurring  among  horses  in  Japan.  Sanfelice  ^ 
has  isolated  similar  microorganisms  from  the  lymph  glands  of  a  horse 
which  was  apparently  suffering  from  a  primary  carcinoma  of  the  liver. 
The  same  author  has  described  a  member  of  this  group  which  he  obtained 
from  a  cheesy  consolidation  occurring  in  the  lung  of  a  hog. 

Demonstration  of  the  organisms  offers  little  difficulty  either  in  fresh 
preparations  of  the  pus  under  a  cover-slip,  or  in  smears  stained  with 

.  1  Rixford  and  Gilchrist,  Johns  Hopkins  Hosp.  Rep.,  i,  1896. 
2  Ophiils,  loc.  cit. 

8  Sanfelice,  Cent.  f.  Bakt.,  I,  xxxi,  1902. 
•  Tokishige,  Cent.  f.  Bakt.,  I,  xix,  1896. 
5  Sanfelice,  Cent.  f.  Bakt.  I,  xviii,  1895,  and  Zeitschr.  f.  Hyg.,  xxi,  1895. 


^ 


THE  YEASTS  633 

thionin,  methylene-blue,  or  the  polychrome  stains.  In  fresh  prepara- 
tions the  addition  of  a  little  NaOH  in  weak  solution  facilitates  the 
search.  When  found,  the  organisms  are  easily  recognized  by  their  size, 
their  highly  refractive  doubly-contoured  cell-membrane,  their  vacuolated 
protoplasm,  and,  most  important,  by  the  discovery  of  budding  forms. 
In  tissue  sections  they  are  recognizable  by  the  ordinary  hematoxylin- 
eosin  technique,  but  may  be  better  demonstrated  by  the  Loeffler  methy- 
lene-blue method  in  use  for  bacterial  tissue-staining.  Excellent  prep- 
arations are  obtained  by  staining  frozen  sections  with  thionin,  a  method 
well  adapted  for  the  demonstration  of  capsules. 

The  cultivation  of  the  blastomycetes  is  comparatively  easy  after 
they  have  once  been  obtained  in  pure  culture.  Their  isolation,  however, 
usually  is  difficult  when  they  occur  in  material  contaminated  with  bac- 
teria. Growing  more  slowly  than  the  bacteria,  cultures  taken  from  such 
contaminated  material  usually  show  very  few  yeast  colonies.  No  special 
methods  of  facilitating  the  procedure  have  been  devised,  but  success 
will  often  attend  painstaking  and  oft-repeated  plating  of  the  mixed 
cultures  in  high  dilution.  The  most  favorable  medium  for  this  process  is 
glucose  agar.  When  once  obtained  in  pure  culture,  the  blastomycetes 
can  readily  be  kept  alive  for  indefinite  periods  by  transplantation  re- 
peated every  two  or  three  months.  On  agar  or  glucose  agar,  colonies 
appear  after  about  three  or  four  days  as  minute,  glistening,  white 
hemispherical  spots  which  are  not  unlike  colonies  of  staphylococcus 
albus.  Planted  in  agar  stab  cultures,  the  microorganisms  indicate 
their  preference  for  a  well-oxygenated  environment  by  growing  but 
slightly  along  the  course  of  the  stab,  but  by  heaping  up  in  a  thick, 
creamy  layer  upon  the  surface  of  the  medium.  This  layer  in  old. 
cultures  may  be  a  quarter  of  an  inch  high.  At  first  white,  the 
growth,  after  three  or  four  weeks,  may  turn  distinctly  yellow  or  even 
brown.  In  broth,  the  microorganisms  form  a  stringy,  gelatinous,  and 
uneven  cloud.  On  Loeffler^ s  blood-serum  media,  and  upon  gelatin, 
growth  is  easily  obtained.  The  gelatin  is  not  liquefied.  Sugar  media 
are  fermented, by  few  of  the  pathogenic  blastomycetes,  a  fact  which 
places  them  rather  in  distinct  contrast  with  the  fermenting  yeasts  used 
in  the  industries. 

Great  and  fundamental  differences  seem  to  exist  between  the  patho- 
genic species  described  by  various  observers,  and  attempts  at  system- 
atizing the  various  members  of  the  group,  such  as  that  by  Rickets,^ 

»  Rickets,  Jour.  Med.  Res.,  6, 1902. 


634  PATHOGENIC  MICROORGANISMS 

have  met  with  almost  insurmountable  obstacles.  Some  of  the  forms 
described,  like  that  of  Busse,  have  fermented  sugars  and  have  not 
formed  mycelia,  while  organisms  like  that  of  Gilchrist  did  not  cause 
fermentation  in  carbohydrate  media,  but,  by  their  formation  of  my- 
celia  under  certain  conditions,  have  indicated  their  close  relationship 
or  possibly  their  identity  with  the  oidia,  transitional  forms  between 
the  yeasts  and  the  hyphomycetes.  It  is  thus,  in  the  light  of  our  pres- 
ent knowledge  of  these  microorganisms,  quite  impossible  to  establish 
within  this  group  a  distinct  classification  that  is  at  all  reliable. 

In  considering  the  possible  origin  of  blastomycotic  infection  in 
animals  and  man,  it  is,  of  course,  important  that  we  should  have  some 
knowledge  as  to  the  pathogenic  properties  of  the  yeast  met  with  and 
handled  in  daily  life.  Rabinowitsch,^  with  this  in  view,  has  investigated 
the  pathogenic  properties  of  fifty  different  species  of  yeasts  obtained 
from  fruit,  manure,  dough,  and  other  sources,  and  among  them  found 
only  seven  varieties  that  had  any  pathogenicity  for  rabbits,  mice,  or 
guinea-pigs.  In  most  of  her  successful  inoculations,  however,  the 
disease  produced  in  the  animals  had  but  very  little  resemblance  to  the 
blastomycotic  conditions  observed  in  man. 

1  Rahirwwitsch,  Zeit.  f.  Hyg.,  xxi,  1895. 


™ 


CHAPTER  XLVI 

HYPHOMYCETES 
{Eumycetes,  Molds)  ' 

The  hyphomycetes  or  molds  interest  the  bacteriologist  chiefly 
because  of  the  frequency  with  which  they  appear  as  contaminants 
during  bacterial  cultivation,  and  because  they  play  the  role  of  incitants 
in  a  few  common  diseases  of  the  skiij  and  mucous  membranes. 

Morphologically  they  are  entirely  distinct  from  and  much  more 
complex  than  the  bacteria.  To  the  yeasts  they  are  more  closely 
related,  the  gap  between  the  two  classes  being  bridged  by  certain 
forms  often  spoken  of  as  "oidia"  which,  though  usually  appearing  in 
the  budding  yeast-form,  may  occasionally  grow  out  in  mycelial  threads. 

The  characteristic  feature  of  the  hyphomycetes  as  a  class  is  the 
formation  of  long,  interlacing  filaments  or  threads,  known  as  mycelia. 
From  these,  branches  come  off  which  are  spoken  of  as  "hyphse."  Each 
mycelial  thread  possesses  a  well-marked,  doubly-contoured  sheath  or 
cell-wall  and  a  finely  granular  protoplasmic  cell-body,  which,  in  some  of 
the  forms,  is  multinucleated. 

Two  main  classes  of  hyphomycetes  are  distinguished,  the  phy  corny - 
cetes,  and  the  so-called  higher  forms,  or  my  corny  cetes.  The  former  class 
is  characterized  by  the  fact  that  no  partitions  exist  within  the  mycelial 
threads  or  hyphse,  the  entire  meshwork  of  a  single  microorganism  con- 
sisting of  one  multinucleated  cell.  This  group,  furthermore,  possesses 
the  power  of  reproduction  by  both  a  sexual  and  an  asexual  process. 
The  second  class,  or  the  mycomycetes,  possess  a  mycehal  meshwork 
which  is  divided  into  numerous  partitions,  and  reproduces  usually  by 
the  asexual  process  only. 

The  process  of  reproduction,  upon  the  basis  of  which  the  separation 
of  groups  within  this  class  is  determined,  is  best  illustrated  by  citing  a 
common  example  of  each  of  the  main  divisions. 

As  an  example  of  the  phycomycetes,  the  division  most  commonly 
met  with  upon  contaminated  gelatin  plates,  or  upon  exposed  and  moist 
organic  matter  of  any  description,  is  the  one  spoken  of  as  the  muco- 

635 


636 


PATHOGENIC  MICROORGANISMS 


rinoB.  Most  forms  belonging  to  this  division  appear,  grossly,  as  a 
light,  cotton-like  fluff,  spreading  in  a  thin  fur  over  the  surface  of  the 
culture  medium.  Examined  with  the  low  power  of  a  microscope,  there 
may  be  seen  a  complicated  network  of  fine  mycelial  threads,  which  show 
no  septa  arid  from  which  delicate  hyphal  branches  arise.  In  the  forma- 
tion of  the  asexual  spore  organs  near  the  tip  of  each  hypha  a  septum 
appears.  The  tip  of  the  hypha  then  gradually  enlarges  and  furms  a 
spherical  capsule  which  is  known  as  the  sporangium.     The  unswoUen 


Fig.  145. — Mucor  mucedo.     Single-celled  mycelium  with   three   hyphae  and 
one  developed  sporangium.    (After  Kny,  from  Tavel.) 


portion  of  the  hypha  which  projects  into  the  sporangium  is  spoken  of 
as  the  columella.  Within  the  sporangium,  a  large  number  of  small, 
round  spores  are  formed.  When  these  are  ripe,  the  wall  of  the  spor- 
angium bursts  and  the  spores  escape.  Upon  suitable  media,  then,  new 
mycelia  develop  from  these  spores.  The  sexual  reproduction,  which  oc- 
curs in  this  group,  takes  place  in  the  following  way:  From  two  hyphae, 
placed  in  close  apposition,  lateral  branches  grow  toward  each  other. 
These  are  spoken  of  as  gametophores.  The  tips  of  the  gametophores 
soon  come  in  contact  and,  for  a  time,  their  protoplasm  freely  inter- 
communicates. Septa  are  then  formed  which  cut  off  from  the  original 
hyphsD  a  central  cell,  the  zygospore.     This  zygospore  gradually  enlarges 


HYPHOMYCETES 


637 


and,  under  favorable  conditions,  sends  out  a  branch  which  terminates 
in  a  non-sexual  sporangium. 

Among  the  higher  molds,  or  mycomycetes,  various  methods  of 
sporulation  occur,  but  sexual  reproduction  does  not  usually  take  place. 

One  of  the  most  commonly  found  genera  is  that  of  Penicillium.  In 
this  form  the  mycelial  threads  are  partitioned  off,  by  many  transverse 
septa,  into  a  number  of  separate  cells.  From  these,  hyphae,  also  sep- 
tate, are  given  off.  From  the  ends  of  these  hyphae,  germinating 
branches  arise  which  are  known  as  conidiophores.  These  conidiophores 
divide  into  two  or  three  septate  branches,  the  sterigmata.     From  the 


Fig.  146. — Mucor  mucedo.  1.  Sporangium,  c.  columella,  m.  sporangium 
sp.  spores.  2.  Columella,  after  bursting  of  sporangium.  3.  Poorly  de- 
veloped sporangia.  4.  Germinating  spore.  5.  Emptying  of  sporangium. .  (After 
Brefeld.) 


ends  of  these,  other  sterigmata  may  be  given  off  and  from  the  tip  of 
each  of  these  a  single  chain  of  spores  or  conidia  are  constricted  off. 
The  result  is  an  appearance  not  unlike  a  hand  in  which  the  wrist 
represents  the  conidiophore;  the  metacarpal  bones,  the  sterigmata;  and 
the  fingers,  the  long  streptococcus-like  chains  of  conidia. 

Similar  to  and  even  more  common  than  the  penicillia  are  the  varieties 
known  as  Aspergillus.  These  forms,  like  the  preceding,  form  delicate 
mycelial  mesh  works  from  which  branches  or  conidiophores  about  3-10 
mm.  in  length,  arise.  These  develop  club-shaped  expaiisions  at  their  free 
ends  and  from  these  club-shaped  expansions  radially  arranged  sterig- 


638 


PATHOGENIC  MICROORGANISMS 


mata  arise.     On  the  ends  of  these  sterigmata  spores  or  conidia  aevelop 
similar  to  those  developed  by  penicillium. 

Other  forms  of  sporulation  occur  within  this  group.  Thus,  tubular 
spore  capsules  may  be  formed  within  the  end  segments  of  the  hyphae, 
known  as  ascospores.  In  other  cases,  within  a  mycelial  thread,  a 
swelling  may  take  place  into  which  protoplasm  flows  from  the  neighbor- 
ing cells,  at  both  ends.     In  this  way,  an  oval  spore  case  is  developed 


«  *  9 

Fig.  147. — Mucor  mucedo.  Formation  of  zygospore.  1.  Two  branches  coa- 
lescing. 2  and  3.  Process  of  conjugation.  4.  Ripe  zygospore.  5.  Germination 
of  zygospore.  6  and  7.  Mucor  erectus.  Azygo  sporulation.  No  two  branches 
meet,  but  form  spores  without  conjugation.  8  and  9.  Mucor  tenuis.  Azygo 
sporulation.  The  spores  grow  out  from  side  branches  without  sexual  union.  (1-5 
after  Brefeld;  6-9  after  Bainier,  from  Tavel.) 


within  the  course  of  the  mycelial  thread.  This  is  known  as  a  chlamy- 
dospore.  The  segments  on  each  side  of  the  chlamydospore  die  out 
and  the  spore  capsule  is  liberated  from  the  mycelium. 

The  classification  of  the  various  divisions  of  the  hyphomycetes  is  a 
problem  requiring  much  study  and  great  botanical  insight,  and  can 
hardly  be  discussed  in  a  general  work  on  bacteriology. 

Upon  artificial  media,  the  members  of  this  group  are  not  at  all 
fastidious^  growing  easily  upon  organic  matter  of  all  kinds,  provided 


HYPHOMYCETES 


639 


moisture  is  present.  In  laboratories  they  are  frequently  found  as  con- 
taminants, and  in  order  to  procure  them  for  purposes  of  study  it  is  only 
necessary  to  expose  agar  or  gelatin  plates  in  a  dusty,  dark  corner.  In 
households  they  are  frequently  found  growing  in  store-rooms  upon  stale 
bread,  shoes,  leather  trunks,  and  on  remnants  of  food.  Most  of  them 
prefer  an  acid  environment  and  are  dependent  upon  a  free  supply  of 
oxygen.  At  room  temperature  they  grow  more  readily  than  at  the 
usual  incubator  temperature. 


DISEASES  CAUSED  B7  HTPHOMYCETES 

Pitjniasis  versicolor  (Microsporon  furfur). — Pityriasis  is  a  disease 
of  the  skin  observed  chiefly  among  persons  living  under  conditions  of 
uncleanliness,  or  among  those  who  combine  these  conditions  with  a 
tendency  to  profuse  perspiration.  It  begins 
as  a  small,  light  brown  or  yellowish  patch 
upon  the  skin  of  the  abdomen,  breast,  or 
back,  is  flat  and  barely  raised  from  the  cuta- 
neous surface.  It  spreads  and  coalesces  with 
similar  spots  until  the  entire  area  resembles 
strongly  the  figures  of  a  map  with  irregular 
continents  and  islands.  The  disease  does 
not  penetrate  into  the  skin  itself,  but  consists, 
as  Plant  has  pointed  out,  of  a  simple  sapro- 
phytism  of  the  inciting  agent  upon  the  skin. 

The  condition  is  caused  by  a  member  of 
the  group  of  Hyphomycetes,  discovered   in 
1846  by  Eichstedt,  and  later  named  Micro- 
sporon furfur.      The  microorganism  consists 
of  a  dense  meshwork  of  mycelial  threads, 
from   which  septate   hyphae   arise   in   large 
numbers.      From  the    ends  of  these,  spores 
arise  in  rows,  after  the  manner  depicted  for 
peniciUium  (Fig.  149).     The  hyphse,  accord- 
ing to  Unna,^  show  a  characteristic  bending  at  right  angles,  due  to 
a  slight  flattening  of  their  diameters.      Characteristic  of  the  micro- 
sporon proper,  in  preparations    made  from  cutaneous  scrapings,  arc 
the  fragments  of  bent  hyphse  and  the  large  numbers  of  free  spores. 


Fig.  148. — Mucor  ramo- 
sus.       Ripe    sporangia    on 
(After  Lindt.) 


columellae. 


1  Unna,  "  Histopathol.  of  Skin,"  transl.,  N.  Y.,  Macmillan,  1896. 
42 


640 


PATHOGENIC  MICROORGANISMS 


Cultivation  of  Microsporon  furfur  has  been  successfully  carried 
out  by  many  observers.^  Growth  is  particularly  characteristic  upon 
potato,  white  or  yellowish-white  colonies  appearing  within  four  or  five 
days  and  rapidly  spreading  over  the  entire  surface  of  the  potato. 

Thrush  {Soor  or  Muguet;  O'idium  albicans). — Thrush  is  a  locaUzed 
disease  of  the  mouth  occurring  most  frequently  in  children  suffering 

from  malnutrition  or  it  occurs,  under  con- 
ditions of  uncleanliness,  upon  an  area  of 
catarrhal  inflammation  of  the  mucous  mem- 
brane. 

The  microorganism  which  causes  the 
condition  was  first  described  by  Langenbeck 
in  1839,  and  has,  since  then,  been  studied 
by  many  observers.  It  was  successfully 
cultivated  by  Grawitz^  in  1886  and  recog- 
nized by  him  as  belonging  to  the  hypho- 
mycetes.  The  most  careful  cultural  studies 
which  have  been  made  upon  the  Oidium 
albicans  are  those  of  Linossier  and  Roux.^ 

Morphologically,  the  oidium  consists  of 
budding  cells,  resembling  those  of  yeast, 
which,  under  certain  conditions,  can  pro- 
duce mycelia  and  hyphae  from  which 
spores  are  developed.  Two  main  varieties 
are  described,  that  which  produces  large 
spores  and  liquefies  gelatin  in  culture,  and 
that  which  gives  rise  to  smaller  spores  and 
fails  to  liquefy  gelatin.  In  many  cases  only 
the  yeast-like  budding  cells  can  be  found; 
these,  however,  when  subjected  to  unfavor- 
able cultural  conditions,  may  be  induced  to 
send  out  hyphae  and  form  spores.  Like 
yeasts,  but  to  a  lesser  degree,  the  Oidium 
albicans  causes  fermentation  in  sugars.  It 
develops  best  under  slightly  acid  conditions 
and  in  the  presence  of  free  oxygen,  upon  gelatin  and  agar. 

Tropical  Sprue. — Sprue  is  a  disease  com-mon  in  the  West  Indies, 


Fig.  149. — Penicillium 
GLAUCUM.  A .  Showing  septate 
mycelia.  B.  End  of  a  hypha 
— branching  into  two  conidio- 
phores,  from  which  are  given 
off  the  sterigmata.  From  the 
ends  of  these  are  developed 
the  round  conidia.  (After 
Zopf.) 


1  Kotjar,  Ref.  Baumgarten's  Jahresbericht,  1892. 

2  Gravyitz,  Virch.  Arch.,  1886. 

'  Linossier  et  Roux,  Comptes  rend,  de  Tacad.  des  sci 


1889. 


HYPHOMYCETES 


641 


Asia,  and  the  South  Sea  Islands.  It  is  characterized  by  recurrent  at- 
tacks of  diarrhea,  which  are  accompanied  in  many  cases  by  a  charac- 
teristic inflammation  of  the  tongue.  It  runs  a  prolonged  course,  but 
is  frequently  fatal.  Ashf ord  ^  has  isolated  from  the  tongue  and  from 
the  stools  of  200  patients  an  organism  which  he  calls  Monilia.  It 
occurs  at  first  as  a  round  yeast-like  body,  which  soon  has  a  tendency 
to  form  mycelia.  It  reproduces  both  by  side  buds  and  by  terminal 
conidia.  It  is  isolated  on  Saboureaud's  4  per  cent  glucose  agar  with  a 
reaction  of  -j-  2  per  cent. 


Fig.  150. — Aspergillus  glaucus.         m.  Mycelial  threads.        s.  Sterigmata, 
r.  Ascospore.     p.  Germinating  conidium.    A.  Ascus.     (After  de  Bary.) 

On  this  medium  it  forms  clean-cut,  round,  hemispherical,  creamy- 
white,  refractive  colonies.  In  gelatin  stabs  it  grows  along  the  line  of 
puncture  with  fine  hair-like  lateral  shoots.  It  does  not  liquefy  gelatin. 
It  turns  milk  alkaline  and  does  not  coagulate  it.  It  produces  acid  and 
gas  on  glucose,  maltose,  levulose,  saccharose,  and  galactose,  but  does 
not  ferment  other  sugar  media.  On  passage  through  laboratory  ani- 
mals (rabbits,  guinea-pigs)  it  produces  a  systemic  mycosis  and  grad- 
ually increases  in  virulence.  With  these,  passage  strains,  stomatitis, 
and  diarrhea  may  be  produced  by  feeding.  Diarrhea  was  also  pro- 
duced in  a  monkey  by  feeding.     The  organism  loses  its  virulence  on 


1  Ashfordy  Am.  Jour,  of  Med.  Sc,  1915,  cl,  p.  680. 


642  PATHOGENIC  MICROORGANISMS 

prolonged    cultivation.      The    etiological    relationship    of    Ashford's 
Monilia  to  Sprue  is  not  as  yet  generally  accepted. 

Favus  (Achorion  Schoenleinii) . — Favus  is  a  disease  attacking  chiefly 
the  hairy  portions  of  the  body  of  man  and  some  domestic  animals. 
In  man,  it  is  found  most  frequently  in  undernourished  children  upon 
the  scalp.  It  is  a  disease  of  extremely  chronic  course  and  is  very  re- 
sistant to  treatment.  Beginning  as  a  small  erythematous  spot,  it  soon 
develops  into  small  sulphur-yellow  cupped  crusts,  which  are  placed 
usually  about  the  base  of  a  hair.  These  may  spread  and  coalesce.  The 
small  indented  crust  is  spoken  of  as  a  scutulum.  When  such  a  scu- 
tulum  is  removed  and  examined  under  a  microscope,  teased  out  in  a 
few  drops  of  strong  sodium  hydrate  solution  (20  per  cent),  the  incitant 

of  the  disease  may  be  easily 
recognized  and  studied.  In  such 
a  preparation  the  center  of  the 
scutulum  is  found  to  be  made 
up  chiefly  of  small  doubly-con- 
toured spores,  which  are  irregu- 
larly oval  or  round,  and  may  be 
arranged  in  chains,  lying  scat- 
tered among  an  extremely  dense 
Fig.  151. — ^Thrush.    Oidium  albicans,  ,  ^       n   n.  v  i 

^  ■    A     /Afx     r/^^        N  meshworkof  fine   mycelial 

unstained.     (After  Zettnow.)  r 

threads.  Toward  the  periphery 
of  the  scutulum,  the  spores  are  less  numerous  and  the  looser  arrange- 
ment of  the  meshwork  permits  us  to  distinguish  distinct  filaments  of 
mycelia  which  give  off  hyph^,  the  ends  of  which  are  often  swollen 
into  small  knobs.  The  interior  of  the  scutulum  usually  contains  a 
pure  culture  of  the  fungus. 

Many  varieties  of  achorion  have  been  described,  but  Plant  ^  believes 
that,  at  the  present  time,  it  is  not  possible  to  separate  these  from  one 
another,  owing  to  the  fact  that  selective  cultivation  has  succeeded  in 
altering  many  of  the  characteristics  displayed  by  many  of  the  strains. 
The  same  observer  recommends  the  following  method  for  obtaining 
pure  cultures  of  this  microorganism.  As  much  of  the  material  as 
can  be  conveniently  obtained  is  gently  rubbed  up  in  a  sterile  mortar 
with  fine  sand  or  infusorial  earth.  The  triturated  material  is  then 
inoculated  into  fluid  agar  and  plates  are  poured. 

Ordinary  streaked  plates  upon  agar  may  also  be  used  with  success 

with  material  directly  from  the  centers  of  scutula. 

1  PUiuty  in  Kolle  und  Wassermann's  "Handbuch,"  I. 


HYPHOMYCETES 


643 


The  achorion  grows  best  upon  acid  agar  at  a  temperature  of 
37.5°  C.  Growth  appears  within  from  forty-eight  hours  to  three  days 
as  yellowish  disks,  which  occasionally  may  be  slightly  furred  with 
aerial  hyphae. 

Ringworm  (Trichophyton  ionsurans)  .-^Umgworm,  Tinea  circinata, 
or  Herpes  tonsurans,  is  a  contagious  disease  of  the  skin  and  hair,  occur- 
ring most  frequently  in  children  and  appearing  upon  the  haired  por- 
tions of  the  body  as  well  as  upon  free  skin.  It  is  characterized  by  the 
formation  of  circular  scaly  patches,  within  which  the  hairs  fall  out. 

The  disease  is  caused  by  several  species  of  the  trichophyton,  a  genus 
of  hyphomycetes.  These  microorganisms  were  first  recognized  as  in- 
citants  of  the  disease  by  Gruby  ^  in  1841,  and  were  studied  later  by 
Sabouraud.2  The  fungi  consist  of  finely  interlaced  narrow  septate 
mycelia,  within  which  characteristic 
swellings  appear.  From  these  swell- 
ings, chlamydospores  develop.  Hyphge, 
both  aerial  and  deep,  grow  out  of  the 
mycelial  threads,  on  the  ends  of  which 
ascospores  may  develop.  In  the  dis- 
eased skin,  the  fungi  grow  within  the 
hair  sheath,  causing  an  area  of  second- 
ary inflammation  about  the  base  of  the 
hair.  The  infection  probably  begins 
first  in  the  epidermis  surrounding  the 
hairs,  from  which  it  then  spreads  into 
the  hair  bulb  and  thence  grows  up  into 
the  substance  of  the  hair  in  which 
mycelial  threads  and  spores  develop  in 
large  numbers.  The  demonstration  of 
the  microorganisms  from  a  case  can  easily  be  accomplished  by  epilating 
an  affected  hair,  making  sure  that  the  hair  bulb  has  been  removed. 
This  is  then  immersed  under  a  cover-slip  in  a  drop  of  sodium  hydrate 
or  potassium  hydrate  solution  and  examined  under  the  microscope.  In 
this  way  enormous  numbers  of  short  mycelial  threads  and  spores  may 
be  seen  to  lie  within  the  bulb.  Many  varieties  of  these  fungi  have 
been  described  from  different  cases.  Their  interrelationship  is  not 
entirely  clear.  In  general,  a  division  is  made  between  those  which 
develop  large  spores  and  a  more  common  small-spored  variety. 

^  Gruby,  Comptes  rend,  de  I'acad.  des  sci.,  13,  1841. 

2  Sabouraud,  Ann.  de  dermal,  et  de  syph.,  3,  1892,  and  4,  1893. 


Fin.  152. — AciioKioN  Schoen- 
leinii. Section  of  faviis  crust.  • 
Stained  by  Gram.  (After 
Fraenkel  and  Pfeiffer.) 


644  PATHOGENIC  MICROORGANISMS 


1 


Cultivation  is  comparatively  simple  and  is  best  carried  out  upon 
acid  glucose  agar  or  gelatin.  Upon  such  media,  within  five  or  six  days, 
mycelial  threads,  which  are  septate  and  form  chlamydospores,  may  be 
observed.  Pigment,  reddish  or  brown,  is  occasionally  noted.  Gelatin 
is  liquefied.  The  disease  may  be  produced  with  such  cultures  upon 
guinea-pigs.  In  man,  the  disease  is  usually  acquired  by  infection  from 
patient  to  patient. 

Other  Diseases  in  which  Hyphomycetes  have  been  Pound. — ^A 
nimiber  of  cases  have  been  described  in  which  members  of  this  group 
have  been  found  at  autopsy  in  the  lungs  of  persons  dying  of  broncho- 
pneumonia.^ In  most  of  these  cases,  the  fungus  found  belonged  to  the 
aspergillus  group  and  was  regarded  as  a  merely  secondary  invader. 
A  few  cases,  however,  have  been  reported  in  which  the  fungus  was  re- 
garded as  the  primary  cause  of  the  disease.  A  single  case  is  on  record, 
in  which  an  intestinal  infection  with  a  member  of  the  genus  mucor 
resulted  in  a  generalized  infection  with  pulmonary  and  secondary 
cerebral  abscesses.  In  birds,  a  disease  of  the  lungs  has  long  been 
known  to  be  due  to  various  species  of  aspergillus.  In  many  domestic 
animals,  diseases  of  the  skin  and  hair  occur  which  are  caused  by  micro- 
organisms similar  to,  or  identical  with,  those  occurring  in  man. 

SPOROTRICHOSIS 

Parasites  closely  allied  to  the  blastomyces  are  the  sporotrices  which 
were  first  described  by  Schenck  ^  in  this  country  and  have  been  very 
thoroughly  studied  by  De  Beurmann  and  Gougerot.  The  parasites 
belong  to  the  Fungi  imperfecta  They  occur  in  lesions  as  oval  or  cigar- 
shaped  spores  (conidia)  and  grow  in  culture  as  branching  septate 
mycelium  with  clusters  of  oval  or  spherical  conidia  about  the  ends  of 
the  hyphse.  According  to  some  observers  the  conidia  are  attached 
to  the  mycelium  by  short  pedicles.  The  conidia  also  occur  along  the 
sides  of  the  hyphse  and  are  often  grouped  in  whorls  about  the  threads. 
Chlamydospores  are  also  found  in  some  cultures.  The  organisms  are 
obligate  aerobes  and  grow  on  all  ordinary  culture  media,  but  better  on 
those  containing  carbohydrates  and  of  slightly  acid  reaction.  The 
growth  forms  a  thick,  leathery,  white  coating  on  the  surface  of  the 
medium  which  in  older  cultures  becomes  coffee-colored,  and  in  some 
instances  black. 

^Sticker,  Nothnagel,  "Spez.  Path.  u.  Ther.,"  14,  1900. 
^Schenck,  Johns  Hopkins  Hosp.  BuU.,  1898,  286. 


HYPHOMYCETES  645 

De  Beurmann  and  Gougerot  ^  have  described  a  number  of  species 
of  sporo trices  which  are  differentiated  by  variations  in  pigment  pro- 
duction, in  optimum  temperature,  and  in  profusion  and  morphology  of 
the  conidia  in  culture.  Other  observers  believe  all  these  organisms 
belong  to  the  same  species.  The  diagnosis  may  be  made  in  some  cases 
by  finding  conidia  in  the  softened  material  from  the  lesions.  These  are 
best  demonstrated  by  Gram's  stain.  In  other  cases  it  is  necessary  to 
resort  to  cultural  methods,  as  the  conidia  can  not  always  be  found  on 
direct  examination. 

Only  a  few  cases  of  the  disease  have  been  reported  in  this  country,  but 
it  is  apparently  common  in  France  and  has  been  reported  in  nearly 
every  quarter  of  the  globe.  The  lesions  are  usually  subcutaneous,  but 
visceral  forms  have  been  described.  Numerous  types  of  lesions  are 
found.  The  commonest  forms  are  disseminated  nodules  which  re- 
semble gummata.  In  other  cases  there  are  scattered  subcutaneous 
abscesses  which  are  usually  associated  with  lymphangitis.  There  is  also  a 
papulo-vesicular  form  which  usually  leads  to  ulceration.  The  lesions 
are  chronic  in  character  and  simulate  the  lesions  of  syphilis  or  tuberculosis, 
for  which  conditions  many  cases  of  sporotrichosis  have  probably  been 
mistaken.  Nodular  lesions  have  also  been  found  in  the  bones,  in 
lymph  nodes,  and  in  the  lungs  and  kidney.  The  lesions  consist  of  foci 
of  chronic  granulation,  the  tissue  containing  numerous  giant  cells, 
which  later  undergo  separation.  There  is  as  a  rule  little  systemic 
disturbance  associated  with  the  disease.  The  lesions  often  heal  spon- 
taneously, leaving  dense  scars,  but  clear  up  very  rapidly  under  iodide 
therapy. 

The  most  susceptible  laboratory  animals  are  mice  and  rats  which, 
show  lesions  resembling  those  in  man  associated  with  marked  cachexia, 
though  the  disease  is  seldom  fatal.  The  disease  has  also  been  produced 
in  rabbits,  guinea-pigs,  and  dogs,  though  these  animals  are  not  sus- 
ceptible to  all  strains.  In  making  cultures  De  Beurmann  and  Gougerot 
recommend  the  use  of  Sabouraud's  glucose  pepton  agar  (water,  1,000  c.c; 
pepton,  10  gm. ;  glucose,  40  gm. ;  agar,  18  gm. ;  not  neutralized) .  Taylor  ^ 
recommends  glycerin  agar  with  the  addition  of  dextrose  and  1  per  cent 
acetic  or  citric  acid  as  the  most  favorable  medium  for  these  organisms. 
Tubes  should  be  inoculated  with  large  amounts  of  pus  (1  c.c.  if  pos- 
sible), and  should  be  incubated  for  several  days  at  room  temperature. 

1  De  Beurmann  et  Gougerot,  "  Traits  des  Sporotrichoses,"  FoUx  Alcan,  Paris,  1912. 

2  Taylor,  Jour.  A.  M.  A.,  1913,  Ix,  1142. 


SECTION  IV 

EXANTHEMATA  AND  DISEASES  CAUSED 
BY  FILTRABLE  VIRUS 


CHAPTER  XLVII 

RABIES 

(Hydrophobia,  Rage,  Lyssa,  Hundswuth) 

Rabies  is  primarily  a  disease  of  animals,  infectious  for  practically 
all  the  mammalia,  but  most  prevalent  among  carnivora,  dogs,  cats,  and 
wolves.  It  is  said  also  to  occur  spontaneously  among  skunks  of  the 
Southwestern  United  States,  and  is  readily  inoculable  upon  guinea-pigs, 
rabbits,  mice,  rats,  and  certain  birds,  chicken  and  geese  being  especially 
susceptible.  Man  is  subject  to  the  disease.  Infection  usually  occurs  as 
a  consequence  of  the  saliva  of  rabid  animals  gaining  entrance  to  wounds 
from  bites  or  scratches.  The  disease  is  prevalent  to  an  alarming  extent 
in  all  civilized  countries  except  England,  where  the  careful  supervision  of 
dogs,  enforcement  of  muzzling  laws,  and  rigid  legislation  regarding  the 
importation  of  dogs,  have  caused  a  practical  eradication  of  the  disease 
in  that  country.  A  fair  estimate  of  the  prevalence  of  the  disease  may 
be  obtained  from  the  statistics  of  animals  dying  or  killed  because  of 
rabies  in  different  countries.  In  Germany,  according  to  Kolle  and 
Hetsch,  during  the  15  years  ending  in  1901,  there  were  9,069  dogs,  1,664 
cattle,  191  sheep,  110  horses,  175  hogs,  79  cats,  16  goats,  1  mule,  and  1 
fox  affected  with  rabies.  In  eastern  United  States  the  disease  is  not  un- 
common. The  statistics  of  the  New  York  Department  of  Health,  for  a 
period  of  six  months  ending  Dec.  31,  1907,  show  74  cases  of  rabies  among 
dogs  in  New  York  City  and  vicinity.  Among  human  beings  the  disease 
is  no  longer  common  in  civilized  countries,  since  early  preventive  treat- 
ment is  successfully  applied  in  almost  all  infected  subjects. 

Experimental  infection  in  susceptible  animals  is  best  carried  out  by 
injections  of  a  salt-solution  emulsion  of  the  brain  or  spinal  cord  of  an 

646 


RABIES  647 

afflicted  animal,  subdurally,  through  a  trephined  opening  in  the  skull, 
but  may  also  be  accomplished  by  injection  into  the  peripheral  nerves, 
the  spinal  canal,  or  the  anterior  chamber  of  the  eye.  Intravenous  and 
intramuscular  injections  are  also  successful,  though  less  regularly  so. 

The  time  of  incubation  after  inoculation  varies  with  the  nature  of  the 
virus  used,  the  location  of  the  injection,  and  the  quantity  injected.  In 
accidental  infections  of  man  and  animals  the  incubation  is  shortest  and 
the  disease  most  severe  when  the  wounds  are  about  the  head,  neck,  and 
upper  extremities  and  are  deeply  lacerated.  This  is  explained  by  the 
fact  that  the  poison  is  conveyed  to  the  central  nervous  system  chiefly 
by  the  path  of  the  nerve  trunks.  This  has  been  experimentally  shown 
by  di  Vestea  and  Zagari  ^  who  inoculated  animals  by  injection  into 
peripheral  nerves,  and  showed  that  the  nerve  tissue  near  the  point  o( 
inoculation  becomes  infectious  more  quickly  than  the  parts  higher  up; 
thus  the  lumbar  cord  of  an  animal  inoculated  in  the  sciatic  nerve  is  in- 
fectious several  days  before  virus  can  be  demonstrated  in  the  medulla. 

In  man,  infected  with  "  street  virus,"  that  is,  with  the  virus  of  a  dog 
or  other  animal  not  experimentally  inoculated,  the  incubation  period 
varies  from  about  forty  to  sixty  days.  Isolated  cases  have  been  reported 
in  which  this  period  was  prolonged  for  several  months  beyond  this. 

The  virulence  of  rabic  virus  may  be  markedly  increased  or  diminished 
by  a  number  of  methods.  By  repeated  passage  of  the  virus  through 
rabbits,  Pasteur^  was  able  to  increase  its  virulence  to  a  more  or  less 
constant  maximum.  Such  virus  which  had  been  brought  to  the 
highest  obtainable  virulence,  he  designated  as  "virus  fixe.'^  Inocu- 
lation of  rabbits,  dogs,  guinea-pigs,  rats,  and  mice  with  such  virus 
usually  results  in  symptoms  within  six  to  eight  days.  The  same  animals 
inoculated  with  street  virus  may  remain  apparently  healthy  for  two  to- 
three  weeks. 

In  dogs  and  guinea-pigs  inoculation  usually  results  first  in  a  stage 
of  increased  excitability,  restlessness,  and  sometimes  viciousness.  This 
is  followed  by  depression,  torpor,  loss  of  appetite,  inability  to  swallow, 
and  finally  paralysis.  In  rabbits  the  disease  usually  takes  the  form  of 
what  is  known  as  "  dumb  rabies,"  the  animals  gradually  growing  more 
somnolent  and  weak,  with  tremors  and  gradual  paralysis  beginning  in 
the  hind  legs. 

In  experimentally  infected  birds  the  disease  is  slow  in  appearing  and 

1  di  Vestea  and  Zagari,  Ann.  de  Tinst.  Pasteur,  iii. 

2  Pasteur's  work  on  rabies.  Compt.  rend,  de  Tacad.  des  sciences,  1881, 1882, 1884, 
1885,  1886,  and  Ann.  de  Tinst.  Pasteur,  1887  and  1888. 


648  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

may  show  a  course  of  gradually  increasing  weakness  and  progressive 
paralysis  extending  over  a  period  of  two  weeks  after  the  appearance  of 
the  first  symptoms. 

In  man,  the  disease  begins  usually  with  headaches  and  nervous  de- 
pression. This  is  followed  by  difficulty  in  swallowing  and  spasms  of  the 
respiratory  muscles.  These  symptoms  occur  intermittently,  the  free 
intervals  being  marked  by  attacks  of  terror  and  nervous  depression. 
Occasionally  there  are  maniacal  attacks  in  which  the  patient  raves  and 
completely  loses  self-control.  Finally,  paralysis  sets  in,  ending  event- 
ually in  death. 

Pathological  examination  of  the  tissues  of  rabid  animals  and  human 
beings  reveals  macroscopically  nothing  but  ecchjonoses  in  some  of 
the  mucous  and  serous  membranes.  Microscopically,  however,  many 
abnormal  changes  have  been  observed  and  were  formerly  utilized  in 
histological  diagnosis  of  the  condition.  Babes  ^  has  described  a  disap- 
pearance of  the  chromatic  element  in  the  nerve  cells  of  the  spinal  cord. 
This  observation  has  been  confirmed  by  others,^  but  is  no  longer  regarded 
as  pathognomonic  of  rabies.  The  same  observer  has  described  a 
marked  leucocytic  infiltration  which  occurs  about  the  blood-vessels  of 
the  brain  and  about  the  ganglia  of  the  sympathetic  system.  These 
changes  are  not  found  in  animals  infected  with  virus  fixe  and  are  present 
only  in  animals  and  human  beings  inoculated  with  street  virus. 

In  1903,  Negri  ^  of  Pa  via  described  peculiar  structures  which  he 
observed  in  the  cells  of  the  central  nervous  system  of  rabid  dogs.  While 
present  in  all  parts  of  the  brain,  these  *' Negri  bodies"  are  most  regularly 
present  and  numerous  in  the  larger  cells  of  the  hippocampus  major  and 
in  the  Purkinje  cells  of  the  cerebellum.  The  presence  of  these  structures 
in  rabid  animals  and  man  has  been  confirmed  by  a  large  number  of 
workers  in  various  parts  of  the  world,  and  the  specific  association  of  these 
bodies  with  the  disease  is  now  beyond  doubt.  In  consequence,  the 
determination  of  "Negri  bodies''  in  the  brains  of  suspected  animals  has 
become  an  extremely  important  method  of  diagnosis — ^more  rapid  and 
accurate  than  the  methods  previously  known. 

The  demonstration  of  Negri  bodies  in  tissues  is  carried  out  as  follows: 
A  small  piece  of  tissue  is  taken  from  the  cerebellum  or  from  the  center 
of  the  hippocampus  major  (cornu  ammonis),  and  is  fixed  for  twelve 
hours  in  Zenker's  fluid.     It  is  then  washed  thoroughly  in  water  and 

1  Bahes,  Virch.  Arch.,  110,  and  Ann.  de  I'inst.  Pasteur,  6,  1892. 

2  Van  Gehuchten,  Bull,  de  Tacad.  de  med.  et  biol.,  1900. 
»  Negri,  Zeit.  f .  Hyg.,  xliii  and  xliv. 


RABIES  649 

dehydrated  as  usual  in  graded  alcohols,  embedded  in  paraffin,  and 
sectioned.  The  sections  are  best  stained  by  the  method  of  Mann,  as 
follows: 

The  sections,  attached  to  slides  in  the  usual  way,  are  immersed  in  the  follow- 
ing solution  for  from  twelve  to  twenty-four  hours: 

Methylene-blue  (Gruebler  00),  1  per  cent 35  c.c. 

Eosin  (Gruebler  BA),  1  per  cent 35  c.c. 

Distilled  water 100  c.c. 

They  are  then  differentiated  in: 

Absolute  alcohol 30  c.c. 

Sodium  hydrate,  1  per  cent  in  absolute  alcohol -5  c.c. 

In  this  solution  blue  is  given  off  and  the  sections  become  red.  After  about  five 
minutes  the  sections  are  removed  from  this  solution,  are  washed  in  absolute  alco- 
hol, and  are  placed  in  water  where  they  again  become  faintly  bluish.  It  is  of  ad- 
vantage to  immerse  them,  now,  in  water  sUghtly  acidified  with  acetic  acid.  Follow- 
ing this  they  are  dehydrated  with  absolute  alcohol  and  cleared  in  xylol,  as  usual. 

In  preparations  made  in  this  way,  the  nerve  cells  are  stained  a  pale 
blue,  and  in  their  cytoplasm,  lying  either  close  to  the  nucleus  or  near  the 
root  of  the  axis-cylinder  process,  are  seen  small  oval  bodies  stained  a  deep 
pink.  The  bodies  are  variable  in  size,  measuring  from  1  to  27  micra  in 
diameter.  They  are  round  or  oval,  show  a  more  deeply  stained  periph- 
eral zone  which  has  been  interpreted  as  a  cell  membrane,  and,  in  their 
interior,  often  show  small  vacuole-like  bodies.  There  may  be  more  than 
one,  often  as  many  as  three  or  four,  in  a  single  cell. 

The  rapid  demonstration  of  Negri  bodies  in  smears  of  brain  tissue 
has  recently  been  advocated  by  many  observers  and  has  been  extensively 
used  for  diagnosis.  It  is  carried  out,  according  to  Van  Gieson,^  in  the 
following  way:  A  small  pin-head-sized  piece  of  brain  tissue  from  the 
regions  indicated  above,  is  placed  on  one  end  of  a  sUde  under  a  cover- 
glass  and  the  cover  is  gently  squeezed  with  the  finger  until  the  tissue  is 
flattened  out  into  a  thin  layer.  The  glass  cover  is  then  gently  shifted 
across  the  slide  until  the  brain  tissue  is  smeared  along  the  entire  surface. 
These  smears  may  be  fixed  in  methyl  alcohol  and  stained  by  the  Giemsa 
method,  as  described  in  the  chapter  on  Spirochseta  pallida  (see  page 
592). 

Stained  in  this  way,  the  Negri  bodies  are  stained  light  blue,  in  con- 
trast to  the  darker  and  more  violet  cell-bodies. 

1  Van  Gieson,  Proc.  of  N.  Y.  Pathol.  Soc,  N.  S.,  iv,  1906. 


650  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

The  smears  may  also  be  stained  by  a  method  originated  by  Van 
Gieson,  which  gives  an  excellent  contrast  stain  and  reveals  more  clearly 
the  inner  structure  of  the  Negri  bodies.  Van  Gieson's  stain  is  prepared 
as  follows : 

Distilled  water 10  c.c. 

Saturated  alcoholic  solution  of  rosanilin  violet 2  drops. 

Saturated  aqueous  solution  of  methylene-blue  diluted  one-half 

with  water 2  drops. 

This  method  has  been  modified  by  Wilhams  and  Lowden/  who  add 
to  10  c.c.  of  distilled  water  3  drops  of  saturated  alcoholic  basic  fuchsin 
and  2  c.c.  of  Loeffler's  methylene-blue.  The  slides  are  fixed  in  methyl 
alcohol,  washe'd  in  water,  and  covered  with  the  freshly  prepared  stain. 
The  sUde  is  held  over  the  flame  until  the  solution  steams  and  is  then 
rinsed  in  water  and  dried.  The  Negri  bodies  assume  a  brilliant  hue  and 
contain  in  their  interior  darkly  stained,  irregular  particles  which  have 
been  interpreted  as  chromatin  bodies.  As  to  the  nature  of  the  Negri 
bodies  opinions  are  still  divided.  Their  constant  presence  in  rabic 
brain  tissue  is  unquestioned  and  their  diagnostic  significance  well 
established.  Cultivation  experiments,  however,  have  been  uniformly 
unsuccessful.  A  number  of  observers,  Negri  himself.  Calkins,^  Williams 
and  Lowden,^  and  others,  beUeve  these  bodies  to  be  protozoa.  The 
last-named  authors  base  this  opinion  upon  the  definite  morphology  oi 
the  bodies,  and  their  staining  properties,  which  in  many  respects  art' 
similar  to  those  of  protozoa.  These  observers  also  claim  that  the  ilior- 
phology  of  the  bodies  shows  a  number  of  regular  cyclic  changes  which 
are  found  accompanying  different  stages  of  the  disease;  these  changes 
correspond,  according  to  these  workers,  to  similar  cycles  occurring 
among  known  protozoa  of  the  suborders  of  the  class  Sporozoa.  Many 
pathologists  still  look  upon  them  as  specific  degenerations  of  the  nerve 
cells  similar  to  the  changes  observed  by  Babes. 

It  is  not  possible  to  decide  absolutely  from  the  facts  at  present  at  our 
disposal  whether  or  not  the  Negri  bodies  should  be  regarded  as  parasites 
or  as  specific  degeneration  products.  Their  constant  presence  in  rabic 
animals,  and  their  apparent  absence  from  normal  brains  and  the  brains 
of  animals  dead  of  other  diseases,  would  tend  to  favor  the  parasitic 
view.  To  us  it  would  seem  that  added  to  this  the  clear  outlines,  apparent 
regularity  of  structure,  and  curiously  consistent  grouping  of  the  darkly 

*  Williams  and  Lowden,  Jour.  Inf.  Dis.,  3,  1906. 

2  Calkins,  Discussion,  Proc.  N.  Y.  Pathol.  Soc,  N.  Si,  vol.  vi,  1906. 

*  WiUiams  and  Lowden,  loc.  cit. 


1 


RABIES  651 

staining  inclusions  would  add  weight  to  such  an  assumption.  We  have 
triturated  rabic  tissue  and  shaken  it  up  in  anti-formin  and  seen  many- 
free  Negri  bodies  apparently  enucleated  from  the  cells  in  consequence. 
Such  complete  extrusion  from  the  cell  also  is  seen  in  the  ordinary  smear 
preparations.  It  is  at  least  unlikely  that  a  cell-degeneration  area  would 
be  expelled  from  the  cytoplasm  in  so  clearly  outlined  and  morphologically 
unaltered  a  form.  The  fact  that  the  virus  is  filtrable,  as  shown  by 
Remlinger,^  Poor  and  Steinhardt,^  and  others,  would  on  the  other  hand 
seem  to  contradict  the  etiological  importance  of  the  Negri  bodies  unless, 
with  some  of  the  observers  named,  we  assumed  them  to  represent  a 
large  stage  in  the  life-cycle  of  a  protozoan  parasite,  which  also  occurred 
in  smaller  forms.  It  is  a  curious  fact,  also,  that  Negri  bodies  are 
scarce  or  absent  in  the  spinal  cord  and  cerebrum,  though  these  areas  are 
as  virulent  or  more  so  than  the  hippocampus  and  cerebellum.  They 
are  small  and  hard  to  find  in  virus  fixe,  largest  and  most  plentiful  in  cases 
in  which  the  incubation  period  has  been  prolonged — as  with  street-virus 
infection.  Much  can  be  said  on  both  sides,  but  in  analyzing  the  present 
experimental  facts,  it  seems  fair  to  say  that  neither  point  of  view  is  cer- 
tain, though  the  parasitic  nature  of  the  Negri  bodies  seems  very  likely. 

.The  cultivation  of  parasites  from  rabic  tissues  has  of  course  been 
attempted  by  most  bacteriologists  who  have  studied  the  disease  since 
Pasteur.  In  all  attempts,  until  very  recently,  either  no  results  were 
obtained  or  else  the  parasites  described  could  be  shown  to  be  pres- 
ent because  of  extraneous  contamination.  Recently  Noguchi  an- 
nounced that  he  has  been  able  to  cultivate  the  virus  by  employing  a 
technique  similar  to  his  methods  of  cultivating  Treponema  pallidum  and 
poliomyelitis  virus.  Into  high  tubes  filled  with  ascitic  fluid  a  bit  of  fresh ' 
sterile  rabbit  kidney  and  a  small  piece  of  rabic  virus  were  placed.  The 
ascitic  fluid  was  covered  with  sterile  oil  and  the  tubes  incubated  at 
37.5°  C  After  five  days'  incubation  cloudiness  appeared  and,  with  it, 
minute  globoid  bodies  not  unlike  those  seen  in  poliomyelitis.  After 
several  generations  large  highly  refractile  bodies  with  dark  central 
spots  appeared  in  the  cultures,  and  these  Noguchi  ^  regards  as  possibly 
the  parasites  and  similar  to  Negri  bodies.  Opinions  are  still  divided  as 
to  the  significance  of  Nogiichi's  results.  However,  whatever  may  be 
one's  opinion  regarding  the  nature  of  the  peculiar  bodies  visible  in  his 
cultures,  he  has  accomplished  the  feat  of  preserving  the  virulence  of  the 

^  Remlinger,  Ann.  de  I'inst.  Past.,  xvii,  1903. 
;,.    2  Poor  and  Steinhardt,  Jour,  of  Inf.  Dis.,  xii,  1913. 
<i'     ^iVo^t^Ai,  Jour.  Exp.  Med.,. xviii,  1913. 


n 


t62  DISiEASES  CAUSED  BY  FILTRABLE  VIRUS 


virus  through  21  generations  on  artificial  media,  a  fact  which  alone  would 
seem  to  prove  that  he  had  successfully  cultivated  it,  even  though  his 
"nucleated  bodies"  do  not  eventually  turn  out  to  be  anything  more 
than  cell  degenerations.  The  possibihty  that  he  may  have  carried 
original  virus  through  21  generations  and  that  it  has  remained  virulent 
for  about  100  days  at  37.5°  C.  can  not  be  excluded  as  yet,  but  seems 
very  remote. 

The  Specific  Therapy  of  Rabies. — The  treatment  which  is  now  pro- 
phylactically  appUed  to  patients  infected  with  or  suspected  of  infection 
with  rabies  has  been  but  little  altered  either  in  principle  or  in  technical 
detail  since  it  was  first  worked  out  by  Pasteur.  In  principle  it  con- 
sists of  an  active  immunization  with  virus,  attenuated  by  drying,  admin- 
istered during  the  long  incubation  period  in  doses  of  progressively 
increasing  virulence. 

By  the  repeated  passage  of  street  virus  through  rabbits,  Pasteur 
obtained  a  virus  of  maximum  and  approximately  constant  virulence 
which  he  designated  as  virus  fixe.  By  a  series  of  painstaking  experi- 
ments he  then  ascertained  that  such  virus  fixe  could  be  gradually  at- 
tenuated by  drying  over  caustic  potash  at  a  temperature  of  about  25° 
C,  the  degree  of  attenuation  varying  directly  with  the  time  of  drying. 
Thus,  while  fresh  virus  fixe  regularly  caused  death  in  rabbits  after  six  to 
seven  days,  the  incubation  time  following  the  inoculation  of  dried  virus 
grew  longer  and  longer  as  the  time  of  drying  was  increased,  until  finally 
virus  dried  for  eight  days  was  no  longer  regularly  infectious  and  that 
dried  for  twelve  to  fourteen  days  had  completely  lost  its  virulence. 

The  method  of  active  immunization,  which  Pasteur  used,  consisted 
in  injecting,  subcutaneously,  virus  of  progressively  increasing  viru- 
lence, beginning  with  that  derived  from  cords  dried  for  thirteen  days 
and  gradually  advancing  to  a  strong  virus.  Thus  the  patient  was  im- 
munized to  a  potent  virus  several  weeks  before  the  incubation  time  of 
his  own  infection  had  elapsed.  Pasteur  successfully  proved  the  efficacy 
of  his  method  upon  dogs  and  finally  upon  human  beings,  the  first 
human  case  being  that  of  a  nine-year-old  child — Joseph  Meister. 

Technique  of  Rabies  Therapy. — The  technique  developed  by 
Pasteur  is  still,  in  the  main,  followed  by  those. who  treat  rabies  to-day. 

I.  As  a  preliminary,  it  is  necessary  to  prepare  or  obtain  virus  fixe. 
This  may  generally  be  procured  from  an  established  laboratory  or  may 
be  prepared  independently  by  passing  street  virus  through  a  series  of 
young  rabbits  (weighing  from  700  to  1 ,000  gms.) .     According  to  Hogyes,^ 

^Hogyes,  quoted  from  Kraus  and  Levaditi,  "Handb.,"  etc.,  I. 


RABIES 


653 


the  passage  of  the  virus  through  twenty-one  to  thirty  rabbits,  in  this 
way,  will  reduce  its  incubation  time  to  seven  or  eight  days.  Babes 
claims  to  obtain  a  virus  fixe  more  rapidly  by  passing  the  virus  alter- 
nately through  rabbits  and  guinea-pigs. 

For  purposes  of  inoculation,  virus  is  prepared  by  emulsifying  in 
sterile  salt  solution  pieces  of  the  medulla  or  cerebellum  of  animals  dead  of 


Fig.  153. — Method  of  Drying  Spinal  Cord  of    Rabbit  for  Purposes 

OF  Attenuation. 


a  previous  inoculation.  The  brain  tissue  which  is  not  emulsified  may  be 
preserved  under  sterile  glycerin  in  a  dark  and  cool  place  for  further  use. 

II.  Rabbits  are  inoculated  with  virus  fixe  by  intracranial  injection. 
A  small  incision  is  made  in  the  shaved  scalp  in  the  median  line,  and  the 
skin  is  retracted.  With  a  small  trephine  or  a  round  chisel,  an  opening 
is  made  in  the  skull  in  the  angle  between  the  coronary  and  sagittal  su- 
tures. Through  this  opening  about  0.2  to  0.3  c.c.  of  the  vines  fixe  is  in- 
jected, either  directly  into  the  brain  substance  or  simply  under  the  dura. 

As  soon  as  a  rabbit  so  inoculated  has  died  it  is  autopsied.  The 
animal  before  dissection  should  be  washed  in  a  disinfectant  solution 
— ^lysol  or  carbolic  acid.  The  skin  is  then  removed  and  the  animal, 
lying  on  its  ventral  surface,  is  fastened  to  a  dissecting  board.  The 
spinal  canal  is  then  laid  open  with  a  pair  of  curved  scissors  and 
the  spinal  cord  carefully  removed.  This  is  accomplished  by  cutting 
across  the  cord  in  the  lumbar  region,  and  lifting  this  with  a  forceps 
while  the  nerve  roots  are  divided  from  below  upward. 

The  cord  is  suspended  by  a  sterile  thread  within  a  large  bottle  into 
the  bottom  of  which  pieces  of  potassium  hydrate  have  been  placed. 
The  bottle  is  then  set  away  in  a  dark  room  or  closet,  the  temperature  of 


654 


DISEASES  CAUSED  BY  FILTRABLE  VIRUS 


which  is  regulated  so  as  to  vary  little  above  25°  C.  Bacteriological 
controls  as  to  the  sterility  of  the  cord  should  also  be  made. 

After  a  suitable  period  of  drying,  pieces  of  the  cord  are  prepared 
for  injection.  This  is  done  in  various  ways  at  different  laboratories. 
No  attempt  at  exact  dosage  is  made.  At  the  New  York  Depart- 
ment of  Health  1  cm.  of  the  cord  is  emulsified  in  3  c.c.  of  sterile 
eight-tenths  per  cent  salt  solution,  the  dose  for  injection  being  usu- 
ally 2.5  c.c.  Marx^  emulsifies  1  cm.  of  the  cord  in  5  c.c.  of  sterile 
bouillon  or  salt  solution,  using  1  to  3  c.c.  of  this  for  injection  according 
to  the  age  of  the  cord.  For  shipment  an  addition  of  20  per  cent  of 
glycerin  and  0.5  per  cent  of  carbolic  acid  is  made. 

The  scheme  of  treatment  is  also  subject  to  variations  according  to 
the  individual  customs  of  various  laboratories.  The  following  scheme 
is  the  routine  of  the  Pasteur  Institute  in  Paris,  as  quoted  in  Kraus 
and  Levaditi,  "Handbuch  fiir  Immunitatsforschung,"  Vol.  I,  p.  713. 


Day  of 
Treatment. 

Mild  Cases. 

Medium  Cases. 

Severe  Cases. 

Days  of 
Drying. 

Dose. 

Days  of  Drying. 

Dose. 

Days  of  Drying. 

Dose. 

1 

14  +  13 

3  c.c. 

14+13 

3  c.c. 

A.M.14+13P.M.12  +  11 

3  c.c. 

2 

12  +  11 

3  c.c. 

12+11 

3  c.c. 

A.M.10  +  9     P.M.   8+    7 

3c.c. 

3 

10  +  9 

3  c.c. 

10  +  9 

3  c.c. 

A.M.         7        P.M.    6 

2  c.c. 

4 

8  +  7 

3  c.c. 

8  +  7 

3  c.c. 

5 

2  c.c. 

5 

6  +  6 

3  c.c. 

6  +  6 

3  c.c. 

5 

2  c.c. 

6 

5 

1  c.c. 

5 

2  c.c. 

•  4 

2  c.c. 

7 

5 

1  c.c. 

5 

2  c.c. 

3 

1  c.c. 

8 

4 

1  c.c. 

4 

2  c.c. 

.    4 

2  c.c. 

9 

3 

1  c.c. 

3 

1  c.c. 

3 

Ic.c. 

10 

5 

2  c.c. 

5 

2  c.c. 

5 

2  c.c. 

11 

5 

2  c.c. 

5 

2  c.c. 

5 

2  c.c. 

12 

4 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

13 

4 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

14 

3 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

15 

3 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

16 

5 

2  c.c. 

5 

2  c.c. 

17 

4 

2  c.c. 

4 

2  c.c. 

18 

3 

2  c.c. 

3 

2  c.c. 

19 

5 

2  c.c. 

20 

4 

2  c.c. 

21 

3 

2  c.c. 

^Marx,  Deut.  med.  Woch.,  1899, 1900. 


RABIES  655 

The  treatment  at  the  New  York  Department  of  Health  is  as  follows:* 


Mild  Cases. 

Medium  Cases. 

Severe  Cases. 

fc 

Day  of 
Treatment. 

Days  of  Drying. 

Dose. 

Days  of  Drying. 

Dose. 

Days  of  Drying. 

Dose. 

1 

14  +  13 

4c.c. 

10 

4  C.C. 

A.M.10  +  9P.M.10  +  9 

4  c.c. 

2 

12+11 

4c.c. 

9 

4  c.c. 

A.M.  8  +  7P.M.  8  +  7 

4  c.c. 

3 

10  +  9 

4  c.c. 

9 

4  c.c. 

6 

4  c.c. 

4 

8  +  7 

4c.c. 

8  +  7 

4  C.c. 

4 

4  c.c. 

5 

6 

2  c.c. 

6 

2  c.c. 

3 

2  c.c. 

6 

5 

2  c.c. 

5 

2  c.c. 

4 

2  c.c. 

7 

4 

2  c.c. 

4 

2  c.c. 

3 

2  c.c. 

8 

3 

2  c.c. 

3 

2  c.c. 

2 

2  c.c. 

9 

5 

2  c.c. 

2 

2  c.c. 

4 

2  c.c. 

10 

4 

2  c.c. 

5 

2  c.c. 

1 

2  c.c. 

11 

3 

2c.c. 

4 

2  c.c. 

4 

2  c.c. 

12 

5 

2  c.c. 

3 

2  c.c. 

3 

2  c.c. 

13 

4 

2  c.c. 

2 

2  c.c. 

2 

2  c.c. 

14 

3 

2  c.c. 

4 

2  c.c. 

4 

2  c.c. 

15 

5 

2  c.c. 

3 

2  c.c. 

1 

2  c.c. 

16 

4 

2  c.c. 

2 

2  c.c. 

4 

2  c.c. 

17 

4 

2  c.c. 

3 

2  c.c. 

18 

3 

2  c.c. 

2 

2  c.c. 

19 

2 

2  c.c. 

4 

2  c.c. 

20 

, 

3 

2  c.c. 

21 

2 

2  c.c. 

22 

4 

2  c.c. 

23 

3 

2  c.c. 

24 

2 

2  c.c. 

25 

4 

2  c.c. 

26 

3 

2  c:c. 

The  severity  or  mildness  of  cases  is  estimated  from  the  depth  and 
degree  of  laceration  of  the  wounds,  also  from  their  location — bites  about 
the  face  and  upper  extremities  being  the  most  dangerous. 

During  the  course  of  such  treatment  patients  may  show  troublesome 
erythema  about  the  point  of  injection  and  occasionally  backache  and 
muscular  pains.  Treatment  need  not  be  omittted  unless  these  symp- 
toms become  excessive. 

The  efficiency  of  the  Pasteur  treatment  in  rabies  is  no  longer  prob- 
lematical. According  to  Hogyes,  50,000  people  have  been  treated  within 
ten  years,  with  an  average  mortality  of  1  per  cent. 


*  Personal  communication  from  Dr.  Poor,  of  the  New  York  Department  of  Health. 
43 


656  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

Although  the  method  described  above  is  the  one  which  is  extensively 
used  in  all  established  institutes  for  the  treatment  of  rabies,  other 
methods  have  been  elaborated  and  used  to  a  slight  extent.  One  of  the 
most  important  of  these  is  the  "dilution  method"  of  Hogyes.  This 
method  is  carried  out  as  follows :  A  definite  quantity  of  the  spinal  cord 
of  a  rabbit  dead  of  virus  fixe  is  emulsified  in  100  c.c.  of  normal  salt 
solution.  Dilutions  of  this  emulsion  are  made  and  the  patient  is  injected 
at  first  with  a  dilution  of  1  :  1,000,  subsequent  injections  being  made  of 
gradually  increasing  concentration  until  a  concentration  of  1  ;  100  is 
reached.  This  method,  so  far  as  it  has  been  used,  has  been  satisfactory, 
but  it  has  not  yet  found  extensive  application. 

Attempts  to  treat  active  rabies  with  the  sera  of  immunized  animals 
have  so  far  been  unsuccessful. 


CHAPTER  XLVIII 
SMALLPOX 

Smallpox  or  variola  is  one  of  the  most  virulent  of  infectious  diseases. 
Throughout  history  it  has  been  a  severe  scourge  of  mankind,  prevailing 
in  China  and  other  Eastern  countries  many  centuries  before  Christ 
and  sweeping  through  medieval  Europe,  especially  at  the  time  of  the 
Crusades,  in  a  series  of  severe  epidemics.  All  races  of  men  are  suscep- 
tible and  no  age  from  childhood  to  senility  is  exempt.  In  modern  times 
the  disease  is  endemic  in  most  uncivilized  countries,  especially  those  of 
the  East,  and  occurs  sporadically  in  all  parts  of  the  globe.  Owing  to 
rigid  enforcement  of  vaccination  and  of  quarantine  laws,  however,  the 
disease  has  been  practically  eradicated  from  civilized  countries. 

The  etiological  factor  which  causes  smallpox  is  still  unknown. 
Numerous  researches  aimed  at  the  discovery  of  cultivatable  microorgan- 
isms in  the  lesions  or  blood  of  infected  patients  have  met  with  uniform 
failure.  Streptococci,  though  often  found  in  the  smallpox  vesicles 
and  pustules,  and  often  undoubtedly  contributing  materially  to  the  fatal 
outcome  of  the  disease,  may  be  regarded  as  purely  secondary  in  signifi- 
cance. 

Communications  which  have  claimed  the  discovery  of  a  protozoan 
incitant  of  the  disease  have,  on  the  other  hand,  been  numerous  and,  in 
some  cases,  have  seemed  plausible.  Yet  absolute  proof  has  always  been 
lacking.  The  literature  on  this  question  is  extensive  and  some  of  the 
earlier  contributions,  such  as  those  of  Griinhagen,^  of  Van  der  Loeff,^ 
and  of  Pfeiffer,^  possess  historical  interest  only.  The  work  which,  of  re- 
cent years,  has  attracted  the  most  serious  attention  to  this  subject  is 
that  published  by  Guarnieri*  in  1892.  This  observer  found,  in  the  deeper 
cells  of  the  epithelium  covering  the  pustules,  both  of  smallpox  lesions 
and  of  vaccination  lesions,  small  bodies  which  were  easily  stained  by 
hematoxylin,  safranin,  or  carmin.     Similar  bodies  could  be  observed  in 

1  Griinhagen,  Arch.  f.  Dermat.  u.  Syph.,  1892. 

2  Van  der  Loeff,  Monat.  f.  prakt.  Dermat.,  iv. 
«  L.  Pfeiffer,  Zeit.  f.  Hyg.,  xxiii. 

*Guamieri,  Arch,  perle  sc.  med.,  xxvi,  1892;  Cent.  f.  Bakt.,  I,  xvi,  1894. 

657 


658  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

the  cells  of  corneal  lesions  experimentally  produced  in  rabbits.  Guar- 
nieri  claimed  that  he  distinguished  both  cytoplasm  and  nucleus  in  these 
bodies  and  described  both  binary  dij/ision  and  reproduction  by  sporu- 
lation  as  in  the  sporozoa.  He  named  the  supposed  protozoan  "Cy- 
toryctes  variolse."  At  about  the  same  time  Monti  ^  described  similar 
bodies  in  the  cells  of  the  Malpighian  layer  of  the  skin  covering  smallpox 
lesions  and,  a  few  years  later,  Clarke  ^  confirmed  the  researches  of 
Guarnieri.  Subsequently,  many  researches  were  carried  out  on  the 
same  subject  in  this  country,  the  most  notable  being  those  of  Council- 
man,^ Magrath  and  Brinckerhoff,  and  of  Calkins/  The  former  authors 
came  to  the  distinct  conclusion  that  the  bodies  seen  by  Guarnieri  were 
parasites,  and  the  latter  author  even  described  a  distinct  life-cycle 
for  these  parasites  comparable  to  that  of  some  protozoa. 

These  researches,  however,  are  by  no  means  absolutely  convincing, 
and  Ewing,^  while  admitting  that  the  vaccine  bodies  are  probablj 
specific  for  variola,  calls  attention  to  the  fact  that  specific  cell-degen- 
erations or  inclusions  are  found  in  diphtheria,  measles,  glanders,  rabies, 
and  other  infectious  processes,  which  can  not  be  regarded  as  in  any 
way  related  to  these  diseases  etiologically,  and  suggests  the  probability 
of  a  similar  interpretation  for  the  vaccine  bodies.  Much  has  been  said 
on  both  sides  of  the  question  since  that  time,  and  the  problem  can  not 
be  regarded  as  settled.  The  burden  of  proof,  of  course,  rests  upon 
those  who  claim  the  discovery  of  a  specific  microorganism,  and  absolute 
proof  will  probably  be  lacking  until  our  experimental  methods  are  such 
as  will  permit  of  other  than  purely  morphological  demonstration. 

Whatever  the  actual  causative  agent  may  be,  it  is  certain  that  the 
disease  is  transmitted  with  extreme  ease — actual  contact,  direct  or  in- 
direct, with  a  patient  being  unnecessary  for  its  transmission.  For  this 
reason  the  disease  is  often  spoken  of  as  being  "air  borne."  While  we 
have  no  certain  knowledge  of  the  portal  of  entry  through  which  the  virus 
invades  the  human  body,  many  considerations  have  made  it  seem  plausible 
that  this  may  take  place  through  the  mucosa  of  the  upper  respiratory  tract. 

Our  knowledge  of  the  means  of  defense  against  the  malady  is  for- 
tunately more  advanced  than  is  that  of  its  etiology.  It  has  been  knovvTi 
for  centuries  that  one  attack  of  smallpox  protects  against  subsequent 
attacks.  This  knowledge  was  made  use  of  by  the  physicians  of  ancient 
China  and  India,  who,  during  mild  epidemics,  exposed  healthy  children 

1  Monti,  Cent.  f.  Bakt.,  I,  xvi.  2  Clarke,  Brit.  Med.  Jour.,  2,  1894. 

3  Councilman,  Magrath,  and  Brinckerhoff,  Jour.  Med.  Res.,  xi,  1904. 

«  Calkins,  Jour.  Med.  Res.,  xi,  1904.  «  Ewin^,  Jour.  Med.  Rerj.,  xiii,  190S.      '■ 


SMALLPOX  659 

to  infection,  hoping  that  mild  attacks  would  result  which  would  confer 
immunity.  While  dangerous  in  the  extreme,  such  "  variolation, '*  never- 
theless, was  not  without  some  benefit  and  was  even  introduced  into 
Europe  in  the  eighteenth  century  by  Lady  Mary  Wortley  Montagu. 

Such  practices,  however,  were  made  unnecessary  by  the  classical 
investigations  of  Jenner  ^  published  in  1798.  Jenner,  as  a  student, 
had  been  impressed  with  the  fact  that  country-people  who  had  been 
infected  with  a  disease  known  as  cowpox,  were  usually  immune  against 
smallpox.  His  studies  arid  observations  came  to  a  practical  issue  when, 
in  1796,  he  inoculated  a  boy,  James  Phipps,  with  pus  from  a  cowpox 
lesion  on  the  hand  of  an  infected  dairy-maid.  Two  months  later  the 
same  boy  was  inoculated  with  material  from  a  smallpox  pustule  without 
subsequent  disease.  With  this  experiment  the  principles  of  vaccination 
as  in  use  at  the  present  time  were  founded. 

The  question  as  to  the  identity  of  cowpox  and  smallpox  has  been 
the  basis  of  a  long  controversy.  Many  obsisrvers  claimed  from  the  be- 
ginning that  the  two  diseases,  though  closely  related  to  each  other,  were 
essentially  different.  Others,  on  thq.  contrary,  and  this  seems  to  be  the 
prevailing  opinion  among  scientists  at  the  present  day,  maintain  that 
cowpox  or  vaccinia,  as  it  is  called  when  inoculated  into  a  human  being, 
represents 'merely  an  altered  and  attenuated  variety  of  variola.  This 
latter  view  is  based  on  the  following  considerations,  which  we  take  from 
Haccius  as  quoted  by  Paul.2 

1.  Variola  is  invariably  transmissible  to  cattle,  when  proper  methods  of  in- 
oculation are  employed. 

2.  Variola  carried  through  several  animals,  in  the  above  way,  becomes  al- 
tered in  character,  approaching  in  nature  typical  vaccinia  or  cowpox. 

3.  Such  virus,  reinoculated  into  man,  gives  rise  to  purely  local  lesions  which 
are  mild  and  unlike  smallpox. 

4.  Inoculation  with  such  virus  protects  both  man  and  animals  against  subse- 
quent inoculation  with  cowpox,  and,  in  the  case  of  man,  against  smallpox  as  well. 

Recently  Kolmer^  has  carried  out  complement-fixations,  using  as 
antigens  salt  solution  suspensions  of  cowpox  and  smallpox  virus,  and 
has  demonstrated  close  biological  relationship  between  the  two. 

It  has  been  claimed,  moreover,  that  cowpox  originally  was  trans- 
mitted to  cattle  by  human  beings  affected  with  smallpox.  This  seems 
likely  both  because  of  the  comparative  rarity  of  the  former  disease 

1  Jenner,  "Inquiry  into  the  Causes  and  Effects  of  the  Variola- Vaccinae,"  London, 
1798.  2  Paid,  "Vaccination";  Kraus  and  Levaditi,  "Handbuch,"  etc.,  l^ 

'  Kolmer,  Jour,  of  Immunology,  No.  1,  Feb.,  1916. 


660  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

and  because  of  its  spontaneous  occurrence  almost  invariably  upon  the 
teats  of  cows,  although  both  males  and  females  are  equally  susceptible 
to  experimental  inoculation. 

The  relationship  of  variola  to  chicken-pox  or  varicella  has  been  more 
easily  determined.  Chicken-pox  does  not  protect  against  smallpox  nor  is 
this  the  case  vice  versa.   The  two  diseases  are  unquestionably  quite  distinct. 

The  Production  of  Vaccine. — During  the  early  days  of  vaccination, 
it  was  customary  to  inoculate  human  beings  with  the  matter  obtained 
from  the  pustules  of  those  previously  vaccinated.  While  this  method 
was  perfectly  satisfactory  for  the  immediate  purposes  in  view,  practical 
difficulties  and  the  occasional  accidental  transmission  of  syphilis  have 
rendered  this  practice  undesirable.  In  consequence,  at  all  institutes 
at  which  vaccine  is  produced  for  use  upon  man,,  the  virus  is  obtained 
from  animals.  Horses  and  mules,  both  extremely  susceptible  to  vac- 
cine, have  been  employed,  and  goats  have,  at  times,  been  chosen  because 
of  their  insusceptibility  to  tuberculosis.  Rabbits  have  also  been  used 
more  recently  by  Calmette  and  Guerin.^ 

The  animals  almost  exclusively.employed  at  the  present  day,  how- 
ever, are  calves,  preferably  at  ages  of  from  six  months  to  two  years. 
Very  young  suckling  calves  are  unsuitable  because  of  the  great  speed  of 
development  and  small  size  of  the  lesions  produced.  The  aninials  should 
be  healthy  and  at  some  institutes  (Dresden)  are  subjected,  before  use, 
to  the  tuberculin  test;  although,  according  to  Paul,^  this  produces  a 
hypersusceptibility  to  the  vaccine,  and  can  be  omitted  without  danger 
when  careful  supervision  is  observed.  Some  observers  prefer  to  use  light- 
colored  animals  rather  than  dark-skinned  or  black  ones,  both  for  reasons 
of  greater  ease  of  cleanliness  and  because  the  former  are  supposed  to  be 
more  susceptible  than  the  latter.  This  contention  is  denied  by  others. 
The  sex  of  the  animals  seems  to  be  immaterial. 

During  the  period  of  use,  the  calves  are  fed,  according  to  age,  with 
either  an  exclusive  milk  diet,  or  they  are  given,  in  addition,  fresh  hay. 
The  greatest  cleanliness  in  regard  to  the  bedding  and  stalls  must  be 
observed  and  separate  stables  should  be  available  for  the  animals  under 
treatment  and  those  under  observation  before  treatment.  These  stables, 
if  possible,  should  be  so  built  that  they  can  be  easily  scoured  and  flushed 
with  water,  and  stalls  should  be  disinfected  after  occupation.  If  possible, 
stables  should  be  artificially  heated  and  a  comfortable  temperature 
maintained.     Halters  and  fastenings  should  be  so  arranged  that  the 

^  Calmette  and  Guerin,  Ann.  de  I'inst.  Pasteur,  1902.  ^  PauL^  loc.  cit. 


SMALLPOX  661 

animals  can  not  lick  the  scarified  surfaces.  Careful  veterinary  control 
before  vaccination  and  during  the  period  of  treatment  must  be  observed 
in  order  to  eliminate  animals  with  systemic  disease  or  other  complications. 

The  calves  may  be  vaccinated  with  material  taken  from  previously 
vaccinated  animals.  Again,  they  may  be  inoculated  with  ''seed  virus" 
obtained  from  the  vesicles  of  human  vaccinia.  This  method  of  using 
humanized  virus  for  the  inoculation  of  calves  for  vaccine  production  is 
preferred  by  many  workers  and  is  spoken  of  as  ''retro vaccination." 

Park  ^  believes  that  the  most  efficient  and  reliable  seed  virus  consists 
of  what  he  calls  human-calf-rabhit  seed  virus.  Crusts  from  healthy 
children,  19  days  after  vaccination,  are  collected.  These  are  cut  up  and 
emulsified  with  boiled  water.  With  this  an  area  of  about  6  inches  square 
on  a  calf,  the  remainder  of  which  is  vaccinated  the  ordinary  way,  is  in- 
oculated. The  virus  from  this  space  is  separately  collected  and,  after 
being  glycerinated,  is  used  in  dilution  of  1  to  123^^  parts  of  salt  solution 
to  vaccinate  rabbits  on  the  shaven  skin  of  the  back.  The  pulp  from  this 
rabbit  vaccination  is  then  used  for  calf  vaccination. 

Actual  vaccination  of  the  animals  is  done  as  follows:  Calves  which 
have  been  kept  under  observation  for  at  least  a  week  are  thoroughly 
washed  and  cleaned  and  the  abdomen  is  clipped  and  shaved  over  an  area 
extending  from  the  ensiform  cartilage  to  the  pubic  region,  including 
the  entire  width  of  the  belly  and  the  inner  folds  of  the  thighs.  It  is 
best  to  shave  the  animal  a  day  or  two  before  vaccination  so  as  to  avoid 
fresh  scratches  and  excoriations.  Just  before  actual  operation  the 
animal  is  strapped  to  a  specially  constructed  operating  table  in  such  a 
way  as  to  allow  free  access  to  the  shaved  area.  This  area  is  now  thor- 
oughly washed  with  soap  and  water  followed  by  alcohol,  or,  in  some 
institutes,  by  a  weak  solution  of  lysol.  If  the  latter  is  used,  the  field 
of  operation  must  again  be  thoroughly  rinsed  with  sterile  water.  About 
a  hundred  small  scarifications  are  made  in  this  area,  preferably  by 
crossed  scratches,  covering  for  each  scarification  an  area  of  about  3-4 
square  centimeters.  Into  these  areas  the  virus  is  rubbed,  using  for  each 
small  area  a  quantity  about  sufficient  to  vaccinate  three  children.  Two 
to  three  centimeter  spaces  are  left  between  the  lesions.  The  lesions  are 
then  allowed  to  dry  and  may  be  covered  with  sterile  gauze  or,  as  in 
Vienna,^  with  a  paste  made  up  of  beeswax,  gum  arable,  zinc  oxid,  water, 
and  glycerin.    In  some  institutes  the  lesions  are  left  entirely  uncovered. 

Ordinarily  within  about  twenty-four  hours  after  vaccination  a  narrow 
pink  areola  appears  about  the  scratches.  Within  forty-eight  hours  the 
1  Park  and  Williams,  Path.  Microorg.,  N.  Y.,  1914,  p.  569.     ^  PaiU,  loc.  cit. 


662  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

scratches  themselves  become  slightly  raised  and  papular,  and  within  four 
or  six  days  typical  vaccinia  vesicles  have  usually  developed. 

To  obtain  the  vaccine  from  such  lesions,  the  entire  operative  field  is 
carefully  washed  with  warm  water  and  soap,  followed  by  sterile  water. 
In  some  cases  two  per  cent  lysol  is  employed,  but  must  again  be  thor- 
oughly removed  by  subsequent  washing  with  sterile  water.  Crusts,  if 
present,  are  then  carefully  picked  off  and  the  entire  contents  of  the  vesi- 
cle, sticky  serum,  and  pulpy  exudate  removed  by  the  single  sweep  of  a 
spoon-curette.  The  curetted  masses  are  caught  in  sterile  beakers  or 
tubes  and  to  them  is  added  four  times  their  weight  of  a  mixture  of  glyce- 
rin fifty  parts,  water  forty-nine  parts,  and  carbolic  acid  one  part.^  Ger- 
man workers  prefer  a  mixture  of  glycerin  eighty  parts,  and  water  twenty 
parts,  omitting  the  use  of  carbolic  acid.  The  glycerinated  pulp  is  allowed 
to  stand  for  three  or  four  weeks  in  order  to  allow  bacteria,  which  are 
invariably  present,  to  die  out.  After  preservation  for  such  a  length  of 
time,  moreover,  thorough  emulsification  is  obtained  more  easily  than 
when  this  is  attempted  immediately  after  curettage.  At  the  end  of 
three  or  four  weeks,  the  glycerinated  pulp  is  thoroughly  triturated, 
either  with  mortar  and  pestle  or  by  means  of  specially  constructed  trit- 
urating devices.  Pulp  so  prepared  should  remain  active  for  at  least  three 
months  if  properly  preserved  in  sealed  tubes  in  a  dark  and  cool  place. 

From  the  serum  oozing  from  the  bases  of  the  lesions,  after  curettage, 
bone  or  ivory  slips  may  be  charged  for  vaccination  with  dry  virus.  The 
glycerinated  pulp  is  put  up  in  small  capillary  tubes,  sealed  at  both  ends, 
and  distributed  in  this  form.  Park  states  that  a  calf  should  yield  about 
10  grams  of  pulp  (which  when  made  up  should  suffice  to  vaccinate  1,500 
persons),  and,  in  addition,  about  200  charged  bone  slips. 

The  virus  may  be  tested  for  its  efficiency  by  a  variety  of  methods. 
Calmette  and  Guerin  ^  inoculate  rabbits  upon  the  inner  surfaces  of  the 
ears  and  estimate  the  potency  of  the  virus  from  the  speed  of  develop- 
ment and  extensiveness  of  the  resulting  lesions.  Guerin^  has  estimated 
the  potency  of  virus  quantitatively  by  a  method  depending  upon  the 
inoculation  of  rabbits  with  a  series  of  dilutions.  Beginning  with  a  mix- 
ture containing  equal  weights  of  glycerin  and  vaccine  pulp,  dilutions  are 
made  with  sterile  water  ranging  from  1  in  10  to  1  in  100.  Rabbits 
are  shaved  over  the  skin  of  the  back  and  1  c.c.  of  each  of  these  dilu- 
tions is  rubbed  into  the  shaved  areas.    Fully  potent  virus  should  cause 

^Huddleston,  quoted  in  Park,  "Pathogenic  Bacteria,"  N.  Y.,  1908. 
*  Calmette  and  Guerin,  Ann.  de  I'inst.  Pasteur,  1902.    • 
3  Guerin,  Ann.  de  I'inst.  Pasteur,  1905. 


SMALL   POX  663 

closely  approximated  vesicles  in  a  dilution  of  1  in  500,  and  numerous 
isolated  vesicles  in  a  dilution  as  high  as  1  in  1,000. 

Quantitative  estimations  of  the  bacteria  in  the  glycerinated  virus 
should  be  made  by  the  plating  method  and  the  vaccine  used  only  when 
after  several  weeks  of  preservation  the  numbers  of  the  bacteria  have 
been  greatly  diminished.  In  glycerinated  pulp  the  bacteria  will  often 
disappear  entirely  in  the  course  of  a  month.  The  vaccine  should  also 
be  tested  for  the  possible  presence  of  tetanus  bacilli,  by  the  inoculation 
of  white  mice.* 

Vaccination  of  human  beings  is  performed  by  slightly  scarifying  the 
skin  of  the  arm  or  leg  with  a  sharp  sterile  needle  or  lancet  and  rubbing 
into  the  lesion  potent  vaccine  virus.  The  virus  was  formerly  dried  upon 
wood,  bone,  or  ivory  slips  and  moistened  with  sterile  water  before  the 
operation.  At  the  present  day  the  glycerinated  pulp  is  almost  univer- 
sally employed. 

That  vaccination  is  of  incalculable  benefit  to  the  human  race  is  no 
longer  a  question  of  opinion,  and  opposition  to  the  practice  is  explicable 
only  on  the  basis  of  ignorance.  Statistical  compilations  upon  this  point 
are  very  numerous.  It  may  suffice  to  select  from  the  voluminous 
literature  a  single  example,  taken  from  Jlirgensen,  which  embodies  the 
statistics  of  death  from  smallpox  in  Sweden,  during  the  periods  immedi- 
ately preceding  and  following  the  introduction  of  vaccination.  In  that 
country  the  first  vaccination  was  done  in  1801.  By  1810  the  practice 
was  generally  in  use  but  not  enforced.  In  1816  it  was  legally  enforced. 
The  years  from  1774  to  1855  can  thus  be  divided  into  three  periods. 

1.  Prevaccinal  period,  1774-1801  (25  years).     Deaths  smallpox  per 

million  inhabitants 2,050 

2.  Transitional  period,  1801-1810  (9  years) .680 

3.  Vaccination  enforced,  1810-1855  (35  years) 169 

Prevaccinal  period  death  rate  20.00  per  mille. 
Vaccinal  period  death  rate         0 . 1 7  per  mille. 

In  considering  the  benefit  of  vaccination  it  must  not  be  forgotten 
that  revaccination  is  quite  as  important  as  the  first  vaccination,  which 
confers  immunity  only  for  from  seven  to  ten  years.  A  child  should  there- 
fore be  vaccinated  soon  after  birth  or  at  least  before  the  eighth  month, 
and  the  process  should  be  repeated  every  seven  years  thereafter. 
- 

» Paid,  loo.  cit. 


CHAPTER  XLIX 
ACUTE  ANTERIOR  POLIOMYELITIS 

The  disease  known  as  acute  anterior  poliomyelitis  has  long  been 
recognized  as  an  acute  infectious  condition,  both  because  of  the  charac- 
teristics of  its  clinical  manifestations  and  of  its  epidemic  occurrence. 
For  these  reasons  it  was  classified  with  acute  infectious  diseases  by 
Marie  and  by  Striimpell  long  before  any  experimental  evidence  of  in- 
fection was  obtainable. 

Its  contagiousness,  while  not  a  proven  fact,  seemed  very  likely  from 
the  evidence  of  its  mode  of  spreading  and  has  been  removed  from  the 
sphere  of  mere  conjecture  by  the  careful  study  of  a  Swedish  epidemic, 
comprising  one  thousand  cases,  made  by  Wickman/ 

While  acute  anterior  poliomyelitis  is  almost  exclusively  a  disease  of 
childhood,  it  is  assumed  by  clinicians  that  it  is  etiologically  closely  re- 
lated to,  possibly  identical  with,  certain  diseases  of  the  adult,  character- 
ized by  bulbar  paralysis  and  acute  encephalitis.  Into  this  category, 
also,  some  observers  place  the  condition  known  as  "  Landry's  paralysis." 
The  basis  for  the  identification  of  these  conditions  with  poliomyelitis 
lies  chiefly  in  the  similarity  of  the  pathological  lesions  and  upon  the 
fact  that  the  last-named  diseases  occur  most  often  during  the  course  of 
poliomyelitis  epidemics. 

In  consequence  of  the  emphatically  expressed  opinion  as  to  the  infec- 
tious nature  of  acute  poliomyelitis,  the  efforts  to  isolate  specific  micro- 
organisms from  cases  have  been  many,  and  numerous  microorganisms 
have  been  described  as  the  causative  agents  of  this  disease.  The  out- 
come of  all  these  investigations  has  been  purely  negative  and  the  infec- 
tious agent  of  acute  poliomyelitis  still  remains  undiscovered.^ 

An  important  advance  in  the  study  of  this  disease  was  made  in  1908 
when  Landsteiner  and  Popper^  succeeded  in  transmitting  it  to  two 
monkeys  ( Cynocephalus  hamadryas  and  Macacus  rhesus).    The  trans- 

»  Wickman,  quoted  from  Landsteiner  and  Popper,  Zeit.  f.  Immunitatsforch,  ^'i, 
1909. 

«  For  literature,  see  Landsteiner  and  Popper,  loc.  cit. 
•Loc.  cit. 

664 


ACUTE    ANTERIOR    POLIOMYELITIS  665 

mission  was  accomplished  by  intraperitoneal  injections  of  a  saline  emul- 
sion of  the  spinal  cord  of  a  child  that  had  died  during  the  fourth  day 
of  an  attack  of  infantile  paralysis — during  the  stage  of  acute  fever.  The 
first  monkey  injected  became  severely  ill  six  days  after  the  injection  and 
died  on  the  eighth  day.  The  second  animal  became  paralyzed  seventeen 
days  after  the  injection  and  was  killed  two  days  later.  Cultural  experi- 
ments with  the  substance  injected  were  negative,  as  were  also  inocula- 
tion experiments  carried  out  upon  guinea-pigs,  rabbits,  and  mice.  The 
histological  lesions  produced  in  the  inoculated  monkeys  were  similar 
to  those  occurring  in  children  afflicted  with  the  disease. 

An  attempt  to  transmit  the  disease  to  another  monkey  with  spinal- 
cord  substance  of  the  animal  that  was  killed  resulted  negatively. 

Soon  after  the  successful  experiments  of  Landsteiner  and  Popper,  a 
similar  result  was  recorded  by  Knoepfelmacher.^  An  attempt  to  trans- 
mit the  disease  from  monkey  to  monkey  was  again  negative. 

Similar  positive  inoculation  results  were  published,  a  little  later  than 
this,  by  Flexner  and  Lewis  ^  in  November,  1909,  and  by  Strauss  and 
Huntoon  ^  in  January,  1910. 

Flexner  and  Lewis,  in  their  work,  moreover,  succeeded  in  trans- 
mitting the  disease  through  several  inoculation-generations  of  monkeys, 
proving  thereby  that  successful  inoculation  did  not  depend  merely 
upon  the  transfer  of  an  unorganized  toxic  body,  but  was  due  to  a  true 
infection.  The  same  workers  *  have  ascertained  that  inoculation  may 
be  successfully  applied  not  only  by  the  intraperitoneal  route  but  intra- 
cerebrally,  subcutaneously,  intravenously,  and  by  the  path  of  the  larger 
nerves.  They  also  proved  that  not  only  the  brain  and  cord  of  afflicted 
animals  contains  the  virus,  but  that  this  may  be  found,  during  the' 
early  days  of  the  disease  at  least,  in  the  spinal  fluid,  the  blood,  the 
nasopharyngeal  mucosa,  and  lymph  nodes  near  the  site  of  inoculation. 

Landsteiner  and  Levaditi,^  meanwhile,  experimenting  with  the  virus 
independently,  succeeded  in  transferring  the  disease  from  one  animal  to 
others,  demonstrated  that  the  virus  could  pass  through  the  pores  of  a 
Berkefeld  filter,  and  showed  that  the  virus  was  present  in  the  salivary 
glands — a  fact  which  may  prove  of  great  importance  in  possibly  estab- 

» Knoepfelmacher,  Mediz.  Klinik,  v,  1909. 

*  Flexner  and  Lewis,  Jour.  Am.  Med.  Assn.,  53, 1909.  ^ 

»  Strauss  and  Huntoon,  N.  Y.  Med.  Jour.,  Jan.,  1910. 
»  Flexner  and  Lewis,  Jour.  Exp.  Med.,  12,  1909. 

» Landsteiner  and  Levaditi,  Comptes  rend,  de  la  soc.  de  biol.,  Nov.,  1909,  and 
Dec.,  1909. 


666  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

lishing  a  clew  to  the  mode  of  contagion  among  human  beings.  The  same 
authors,  as  well  as  Flexner  and  Lewis,  were  able  to  show  that  the  virus 
was  preservable  under  glycerin  for  as  long  as  ten  days  and  retained  its 
virulence  for  from  seven  to  eleven  days  when  dried. 

According  to  Flexner  and  Lewis  the  virus  remains  active,  when 
frozen,  for  as  long  as  forty  days,  but  is  extremely  sensitive  to  heat, 
bling  destroyed  by  a  temperature  of  from  45°  to  50°  C.  maintained  for 
thirty  minutes. 

Experiments  aimed  at  the  isolation  or  even  morphological  detection 
of  a  parasite  in  the  virulent  material  have  been  entirely  without  success 
until  recently.  Bacteria  which  in  the  past  have  been  isolated  from 
nerve  substance  and  spinal  fluid  in  cases  of  poliomyelitis  can  of  course 
be  excluded  from  etiological  significance  by  the  recent  determination  of- 
the  filtrability  of  the  virus  as  determined  by  Flexner  and  Lewis,  and 
Landsteiner  and  Levaditi.  Small  coccoid  forms  in  smears  from  the 
nerve  tissue  recently  described  by  Proescher^  are  of  very  uncertain 
significance.  The  streptococci  recently  described  by  Rosenow  are,  in 
our  opinion,  secondary  invaders.  The  most  important  contribution 
which  has  been  made  in  the  solution  of  this  problem  is  that  of  Flexner 
and  Noguehi.2  These  investigators  placed  small  bits  and  emulsions  of 
the  brain  of  monkeys,  dead  of  poliomyelitis,  in  high  tubes  containing 
human  ascitic  fluid  together  with  a  piece  of  fresh  sterile  rabbit  kidney. 
In  all  essentials  the  method  was  that  followed  by  Noguchi  in  his  culti- 
vation of  Treponema  pallidum.  It  was  necessary  to  use  fresh  unheated 
ascitic  fluid.    Heat  sterilization  rendered  it  unsuitable. 

By  this  method,  after  five  days  opalescence  appeared  about  the 
pieces  of  tissue.  This  increased  until  the  tenth  day,  when  sedimenta- 
tion began.  Microscopical  examination  by  Giemsa's  method  of  stain- 
ing revealed  small  globoid  bodies  measuring  from  0.15  to  0.3  micron  in 
diameter,  arranged  in  pairs,  short  chains,  and  masses.  Sirtiilar  bodies 
could  later  be  found  in  poliomyelitis  tissues.  Cultures  were  obtained 
from  glycerinated  as  well  as  from  fresh  virus  and  from  the  filtered  as 
well  as  the  unfiltered  material.  Typical  lesions  and  death  have  been 
produced  in  monkeys  with  such  cultures  in  a  few  cases. 

We  have  few  data  which  throw  light  upon  possible  immunity  to  the 
disease.  Repeated  attacks  of  the  disease  in  the  same  human  being 
have  not  been  noted ;  but  this,  as  Flexner  and  Lewis  point  out,  may 
be  due  to  the  fact  that  the  epidemics  are  rare,  and  individuals  once 

1  Proescher,  N.  Y.  Med.  Jour.,  1913. 

2  Flexner  and  Noguchi^  Jour,  of  Exp.  Med.,  xviii,  1913. 


ACUTE  ANTERIOR  POLIOMYELITIS  667 

afflicted  have  passed  beyond  the  susceptible  age  by  the  time  of  the  sec- 
ond epidemic.  As  a  matter  of  fact,  however,  these  workers  have  not 
succeeded  in  reinfecting  monkeys  that  had  recovered. 

In  chickens  a  disease  has  been  observed  similar  in  many  ways  to 
poliomyelitis,  but  further  study  has  shown  this  to  be  a  polyneuritis. 

Of  other  animals  besides  monkeys,  rabbits  only  have  been  success- 
fully inoculated  with  this  disease.  Transmission  to  these  animals  was 
first  reported  by  Kraus  and  Meinicke  ^  and  later  by  Lentz  and  Hunte- 
miiller.2  Marks  ^  has  studied  the  disease  in  rabbits  thoroughly,  and 
concludes  that  there  is  no  doubt  that  the  virus  can  be  cultivated 
through  a  limited  number  of  generations  in  rabbits.  He  was  able  to 
transmit  to  monkeys  from  rabbit  material.  The  disease,  however,  does 
not  resemble  that  of  man  or  monkeys  clinically  and  no  definite  lesions 
of  the  central  nervous  system  are  present.  The  rabbits  seem  perfectly 
well  for  six  or  seven  days,  when  rapid  weakness  and  death  in  con- 
vulsions occur. 

Animals  which  have  been  unsuccessfully  injected,  even  tvith  living 
virus,  do  not  develop  immunity.  However,  animals  that  have  been 
successfully  inoculated  and  recovered  are,  like  human  beings,  thereafter 
immune.  Levaditi  and  Landsteiner,  Roemer  and  Joseph,  and  Flexner 
and  Lewis  have  shown  that  the  serum  of  recovered  monkeys  will  pro- 
tect normal  animals  from  fatal  doses  of  the  virus.  That  the  same  pro- 
tective power  for  monkeys  has  been  shown  in  the  serum  of  human 
recovered  cases,  is  shown  by  the  same  authors  and  by  Anderson,  and 
Frost,  consequently  the  intraspinous  injection  of  the  serum  of  recently 
recovered  children  into  patients  in  high  stages  of  the  disease  has  re- 
cently been  advocated  and  is  thought  well  of  by  a  number  of  observers.* 
This  work,  however,  has  not  reached  completion  and  final  judgment 
must  be  withheld. 

*  Kraus  und  Meinicke,  Deut.  med.  Woch.,  xxxv,  1909. 

2  Lentz  und  Huntemuller,  Zeitschr.  f.  Hyg.,  Ixvi,  1910. 

3  Marks,  Jour,  of  Exp.  Med.,  xiv,  1911. 


CHAPTER  L 
YELLOW  FEVER 

Yellow  fever  is  an  acute  infectious  disease  which  prevails  endemi- 
cally  in  the  tropical  countries  of  the  Western  Hemisphere,  but  occurs  also 
along  the  western  coast  of  Africa  and  has  exceptionally  appeared,  in 
epidemic  invasions,  in  the  north  temperate  United  States  and  Europe. 
Guiteras,  as  quoted  by  Osier,  classifies  the  distribution  of  the  disease  into 
three  areas  of  infection. 

1.  The  area  in  which  the  disease  is  never  absent,  including  tropical 
South  American  ports  and  Havana. 

2.  The  area  of  periodic  epidemics,  including  sea-ports  of  the  tropical 
Atlantic  in  America  and  Africa. 

3.  The  area  of  accidental  epidemics,  extending  from  parallel  45° 
north  latitude  to  35°  south  latitude.  In  the  United  States  severe  epi- 
demics have  frequently  occurred  in  Louisiana,  Mississippi,  and  Alabama, 
and  occasional  but  severe  epidemics  have  occurred  in  Philadelphia  and 
Baltimore. 

The  disease  occurs  spontaneously  only  in  man,  and  experimental 
inoculation  of  lower  animals  has  been  successful  only  in  the  chimpanzee 
in  a  single  case  reported  by  Thomas.^ 

In  man  afflicted  with  the  malady  the  clinical  picture  is  one  of  a  rapidly 
developing  fever  with  severe  gastrointestinal  symptoms,  vomiting  of 
blood,  albuminuria,  and  often  active  delirium.  The  mortality  is  usually 
high,  often  reaching  eighty  per  cent  or  more  in  the  severe  epidemics. 

Etiology  and  Method  of  Transmission. — ^The  actual  infective  agent 
which  causes  yellow  fever  is,  as  yet,  unknown.  Numerous  researches 
have  been  aimed  at  the  elucidation  of  the  problem,  and  microorganisms, 
for  which  etiological  significance  was  claimed,  have  been  isolated  from 
the  dejecta,  the  vomitus,  and  the  secretions  of  afflicted  patients.  None 
of  these  claims  has  been  supported  by  convincing  proof  and  none  of 
them  has  found  subsequent  confirmation. 

A  few  of  these  claims  only  have  historical  importance  because  of  the 

I  Thomas,  Brit.  Med.  Jour.,  1,  1907. 
668 


YELLOW  FEVER  669 

widespread  interest  they  aroused  among  bacteriologists.  Comil  and 
Babes/  in  1883,  described  chained  cocci  to  which  they  attributed  etio- 
logical significance,  but  their  contentions  have  remained  entirely  un- 
confirmed. Sternberg,^  in  1897,  described  a  colon-like  organism,  "  bacil- 
lus X,"  for  which  he  made  very  conservative  claims,  which  he  himself, 
later,  withdrew. 

The  most  active  discussion  was  aroused  by  the  announcement  of 
Sanarelli,'  in  1897,  that  he  had  discovered,  in  the  blood  and  tissues  of 
patients  dead  of  yellow  fever,  a  Gram-negative  bacillus,  which  he  be- 
lieved to  be  the  etiological  agent  of  the  disease.  He  based  his  contention 
upon  the  facts  that  he  had  isolated  the  organism  from  seven  cases  of 
yellow  fever,  had  produced  symptoms  similar  to  the  disease  of  the  human 
being  by  the  inoculation  of  pure  cultures  into  dogs,  and  had  obtained 
agglutination  of  the  bacillus  in  the  serum  of  convalescent  patients.  Later' 
he  inoculated  five  human  beings  subcutaneously  with  sterilized  cultures 
of  this  "  Bacillus  icteroides,"  and  obtained  symptoms  which  he  believed 
simulated  closely  those  of  yellow  fever.  The  claims  of  Sanarelli  at  first 
found  much  apparent  confirmation,  but  later  work  by  Durham  and 
Myers,*  Otto,^  Agramonte,*  and  others  has  definitely  refuted  his  original 
claims,  and  there  is  to-day  no  scientific  basis  for  the  assumption  that 
the  Bacillus  icteroides  has  any  etiological  relationship  to  the  disease. 
Protozoan  incitants,  also,  have  been  described  by  Klebs,'  Schiiller,* 
Thayer,^  and  others,  without  bringing  conviction  or  even  justifying 
extensive  investigation  of  their  claims. 

While  thus  the  causative  agent  of  yellow  fever  remains  undiscovered, 
some  of  its  biological  properties  are  known.  Reed,  Carroll,  Agramonte, 
and  Lazear^^  were  able  to  show  that  the  infecting  agent  is  present  in- 
the  blood  serum  of  patients  during  the  first  three  days  of  the  disease 
and  that  it  could  pass  through  the  pores  of  Berkefeld  filters.  Such 
filtered  serum  remained  infectious  for  human  beings — a  fact  which  de- 
monstrates that  the  incitant  is   extremely  small  and  possibly  ultra- 

»  Comil  and  Babes,  Comptes  rend,  de  Tacad.  des  sci.,  1883. 

2  Sternberg,  Cent.  f.  Bakt.,  I,  xxii,  1897. 

»  Sanarelli,  Ann.  de  T  inst.  Pasteur,  1897,  and  Cent,  f .  Bakt. ,  I,  xxii,  xxvii,  and  xxix. 

•  Durham  and  Myers,  Thompson  Yates  Laboratory  Reports  3  1902. 
*Otto,  Vierteljahrsch.  f.  gericht.  Medizin,  etc.,  27,  1904. 

•  Agramonte,  N.  Y.  Med.  News,  1900. 

»  Klebs,  Jour.  Am.  Med.  Assn.,  April,  1898. 
8  Schuller,  Berl.  klin.  Woch.,  7,  1906. 

•  Thayer,  Med.  Record,  1907. 

"  Reedf  Carroll,  Agramonte,  and  Lazear,  Phila.  Med.  Jour.,  1900. 


670  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

microscopic.  Blood  serum,  filtered  or  unfiltered,  becomes  non-infectious 
when  heated  to  56°  C.  for  ten  minutes. 

Mode  of  Transmission. — Until  comparatively  recent  years  the  mode 
of  transmission  of  yellow  fever  was  not  understood  and  many  erroneous 
theories  were  prevalent.  It  was  supposed  that  yellow  fever  was  conta- 
gious, and  transmitted  from  person  to  person  by  direct  or  indirect  con- 
tact with  those  afHicted  or  by  fomites.  The  first  to  make  the  definite 
assertion  that  yellow  fever  was  transmitted  by  the  agency  of  mosquitoes 
was  Carlos  Finlay.  Finlay/  as  early  as  1881,  advanced  the  theory  that 
mosquitoes  w^ere  responsible  for  the  transmission  of  this  disease  and,  fur- 
thermore, recognized  '^  Stegomyia  fasciata  "  or  "  Stegomyia  calopus  "  as 
the  guilty  species.  Finlay 's  opinion,  although  later  proved  to  becorrect, 
was  at  first  based  only  upon  such  circumstantial  evidence  as  the  corre- 
spondence of  the  yellow-fever  zones  with  the  distribution  of  this  species 
of  mosquito  and  the  great  prevalence  of  mosquitoes  at  times  during 
which  epidemics  occurred.  His  theory  was,  therefore,  received  with 
much  skepticism  and  was  neglected  by  scientists  until  its  revival  in 
1900,  when  the  problem  was  extensively  investigated  by  a  commission 
of  American  army  surgeons. 

Reed,  Carroll,  Agramonte,  and  Lazear  were  the  members  of  this 
commission.  The  courage,  self-sacrifice,  and  scientific  accuracy  which 
characterized  the  work  of  these  men  have  made  the  chapter  of  yellow 
fever  one  of  the  most  brilliant  in  the  annals  of  American  scientific 
achievement. 

Their  work  was  much  facilitated  by  the  experience  of  Gorgas  ^  and 
others,  who  had  demonstrated  the  absolute  failure  of  ordinary  sanitary 
regulations  to  limit  the  spread  of  yellow  fever. 

They  began  their  researches  by  investigating  carefully  the  validity 
of  Sanarelli's  claims  as  to  the  etiological  significance  of  his  "Bacillus 
icteroides."  The  results  of  this  work  yielded  absolutely  no  basis  for 
confirmation. 

They  then  proceeded  to  investigate  the  possibility  of  an  intermediate 
host. 

In  August,  1900,  the  commission  began  its  work  on  this  subject  by 
allowing  mosquitoes,^  chiefly  those  of  the  stegomyia  species,  to  suck 

1  Finlay,  Ann.  Roy.  Acad.  d.  Havana,  1881. 

2  Gorgas,  Jour,  of  Trop.  Med.,  1903. 

»  Reed,  Carroll,  Agramonte,  and  Lazear,  Phila.  Med.  Jour.,  Oct.,  1900;  also  Am. 
Pub.  Health  Assn.  Rep.,  1903;  Agramonte,  N.  Y.  Med.  News,  1900;  Reed,  Jour,  of 
Hyg.,  1902;  Reed,  Carroll,  and  Agramonte,  Am.  Medicine,  July,  1901.     Boston  Med. 


Yellow  feIveir 


671 


blood  from  patients,  later  causing  the  same  insects  to  feed  upon  normal 
susceptible  individuals.  The  first  nine  experiments  were  negative. 
The  tenth,  of  which  Carroll  was  the  subject,  was  successful.  Four  days 
after  being  bitten  by  the  infected  insect  Carroll  became  severely  ill  with 
an  attack  of  yellow  fever,  by  which  his  life  was  endangered,  and  from 
the  effects  of  which  he  died  several  years  later. 

On  the  13th  of  September,  Lazear,  while  working  in  the  yellow-fever 
wards,  noticed  that  a  stegomyia  had  settled  upon  his  hand,  and  deliber- 


FiG.  155. — Stegomyia  Fasciata.    (a)  Female,    (b)  Male. 


(W 
(After  Carroll.) 


ately  allowed  the  insect  to  drink  its  fill.  Five  days  later  he  became  ill 
with  yellow  fever  and  died  after  a  violent  and  short  illness. 

With  these  experiences  as  a  working  basis,  the  commission  now 
decided  upon  a  more  systematic  and  thoroughly  controlled  plan  of 
experimentation. 

In  November  of  the  same  year,  1900,  an  experiment  station,  "Camp 
Lazear,"  was  established  in  the  neighborhood  of  Havana,  about  a  mile 
from  the  town  of  Quemados.  The  camp  was  surrounded  by  the  strictest 
quarantine.  Volunteers  from  the  army  of  occupation  were  called  for, 
and  twelve  individuals  were  selected  for  the  camp,  three  immunes  and 
nine  non-immunes.    Two  of  the  latter  were  physicians.    The  immunes 


and  Surg.  Jour.,  14,  1901;  Carroll,  Jour.  Am.  Med.  Assn.,  40, 1903;  Carrol, 
Fever"  in  Mense,  "  Handbuch  der  Tropen-Krankheiten,"  ii. 
44 


YeUo\v 


672  I  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

and  the  members  of  the  commission  only  were  allowed  to  go  in  and  out. 
All  non-immunes  who  left  the  camp  were  prohibited  from  re-entering  and 
their  places  taken  by  other  non-immune  volunteers.  During  December, 
five  of  the  non-immune  inmates  were  successfully  inoculated  with  yel- 
low fever  by  means  of  infected  mosquitoes.  During  January  and  Febru- 
ary five  further  successful  experiments  were  made.  Clinical  observa- 
tions were  made  by  experienced  native  physicians,  Carlos  Finlay  among 
them,  and  the  patients,  as  soon  as  they  were  unquestionably  ill  with 
yellow  fever,  were  removed  to  a  yellow-fever  hospital.  This  was  done 
to  prevent  the  possibility  of  the  disease  spreading  within  the  camp  it^ 
self.  The  mosquitoes  used  for  the  experiments  were  all  cultivated  from 
the  larva  and  kept  at  a  temperature  of  about  26.5°  C. 

A  further  important  experiment  was  now  made  A  small  house  was 
erected  and  fitted  with  absolutely  mosquito-proof  windows  and  doors. 
The  interior  was  divided  by  wire  mosquito  netting  into  two  spaces.  With, 
in  one  of  these  spaces  fifteen  infected  mosquitoes  were  liberated.  Seven 
of  these  had  fed  upon  yellow-fever  patients  four  days  previously;  four, 
eight  days  previously;  three,  twelve  days  previously;  and  one,  twenty- 
four  hours  previously.  A  non-immune  person  then  entered  this  room 
and  remained  there  about  thirty  minutes,  allowing  himself,  to  be  bitten 
by  seven  mosquitoes.  Twice  after  this  the  same  person  entered  the 
room,  remaining  in  it  altogether  sixty-four  minutes  and  being  bitten  fif- 
teen times.   After  four  days  this  individual  came  down  with  yellow  fever. 

In  the  other  room  two  non-immunes  slept  for  thirteen  nights  with- 
out any  evil  results  whatever. 

It  now  remained  to  show  that  mosquitoes  were  the  sole  means  of 
transmission  and  to  exclude  the  possibility  of  infection  by  contact  with 
excreta,  vomitus,  or  fomites.  For  this  purpose  another  mosquito-proof 
house  was  constructed.  By  artificial  heating  its  temperature  was  kept 
above  32.2°  C.  and  the  air  was  kept  moist  by  the  evaporation  of  water. 
Clothing  and  bedding,  vessels,  and  eating  utensils,  soiled  with  vomitus, 
blood,  and  feces  of  yellow-fever  patients  were  placed  in  this  house  and 
three  non-immune  persons  inhabited  it  for  twenty  days.  During  this 
time  they  were  strictly  quarantined  and  protected  from  mosquitoes. 
Each  evening,  before  going  to  bed,  they  unpacked  and  thoroughly 
shook  clothing  and  bedding  of  yellow-fever  patients,  and  hung  and 
scattered  these  materials  about  their  beds.  They  slept,  moreover,  in 
contact  with  linen  and  blankets  soiled  by  patients.  None  of  these 
persons  contracted  yellow  fever.  The  same  experiment  was  twice  re- 
peated by  other  non-immunes,  in  both  cases  with  like  negative  results. 


YELLOW  FEVER  673 

All  of  the  non-immunes  taking  part  in  these  experiments  were 
American  soldiers.  Four  of  them  were  later  shown  to  be  susceptible 
to  yellow  fever  by  the  agencies  of  mosquito  infection  or  blood-injection. 

The  results  obtained  by  the  investigations  of  this  commission  may 
be  summarized,  therefore,  as  follows: 

Yellow  fever  is  acquired  spontaneously  only  by  the  bite  of  the 
Stegomyia  fasciata.  It  is  necessary  that  the  infecting  insect  shall  have 
sucked  the  blood  of  a  yellow-fever  patient  during  the  first  four  or  five 
days  of  the  disease,  and  that  an  interval  of  at  least  twelve  days  shall 
have  elapsed  between  the  sucking  of  blood  and  the  reinfection  of  an- 
other human  being.  Sucking  of  the  blood  of  patients  advanced  beyond 
the  fifth  day  of  the  disease  does  not  seem  to  render  the  mosquito  infec- 
tious, and  at  least  twelve  days  are  apparently  required  to  allow  the  para- 
site to  develop  within  the  infected  mosquito  to  a  stage  at  which  rein- 
fection of  the  human  being  is  possible. 

The  results  of  the  American  Commission  were  soon  confirmed  by 
Guiteras  ^  and  by  Marchoux,  Salimbeni,  and  Simond.^  These  latter  ob- 
servers, moreover,  confirmed  the  fact  that  infection  could  be  experi- 
mentally produced  by  injections  of  blood  or  blood  serum  taken  from 
patients  during  the  first  three  days  of  the  disease.  They  showed  that 
blood  taken  after  the  fourth  day  was  no  longer  infectious:  that  0.1  c.c. 
of  serum  sufficed  for  infection  and  finally  that  no  infection  could  take 
place  through  excoriations  upon  the  skin.  They  furthermore  confirmed 
the  observation  of  Carroll  that  the  virus  of  the  disease  could  pass  through 
the  coarser  Berkefeld  and  Chamberland  filters, — passing  through  a 
Chamberland  candle  "  F  "  but  held  back  by  the  finer  variety  known  as  "  B." 

The  fundamental  factors  of  yellow-fever  transmission  thus  discovered, 
we  are  in  possession  of  logical  means  of  defense.     The  most  important* 
feature  of  such  preventive  measures  must  naturally  center  upon  the 
extermination  of  the  transmitting  species  of  mosquito. 

Stegomyia  fasciata  or  calopus  is  a  member  of  the  group  of  ''  Culi- 
cidae.'^  It  is  more  delicately  built  than  most  of  the  other  members  of 
the  group  cuHcidae,  is  of  a  dark  gray  color,  and  has  pecufiar  thorax- 
markings  which  serve  to  distinguish  it  from  other  species.  The  more 
detailed  points  of  differentiation  upon  which  an  exact  zoological  recog- . 
nition  depends  are  too  technical  to  be  entered  into  at  this  place. 
Briefly  described,   they    consist    of  lyre-Uke   markings   of  the  back, 


»  Guiteras,  Rev.  d.  m^d.  trop.,  Jan.,  1901,  and  Am.  Med.,  11,  1901. 
» Marchoux,  SaUmi?eni,  and  Simond,  Ann.  de  I'inst.  Pasteur,  1903. 


674  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

unspotted  wings,  white  stripes  and  spots  on  the  abdomen,  and  band- 
like white  markings  about  the  metatarsi  and  tarsi  of  the  third  pair 
of  legs.  The  peculiar  power  of  transmitting  yellow  fever  possessed 
by  this  species  is  explained  by  Marchoux  and  Simond  ^  by  the  fact 
that  Stegomyia  fasciata  is  unique  among  culicidae  in  that  the  female 
lives  for  prolonged  periods  after  sucking  blood.  Among  other  species 
— Culex  fatigans,  Culex  confirmatus,  and  most  others — the  female  lays 
its  eggs  within  from  two  to  eight  days  after  feeding  on  blood  and  rarely 
lives  longer  than  the  twelfth  day — the  time  necessary  for  the  develop- 
ment of  the  yellow-fever  parasite. 

The  limitation  of  yellow  fever  to  tropical  countries  ^  is  explained  by 
the  fact  that  stegomyia  develops  only  in  places  where  high  tempera- 
tures prevail.  The  optimum  temperature  for  this  species  lies  between 
26°  and  32°  C.  At  17°  C.  it  no  longer  feeds,  and  becomes  practically 
paralyzed  at  15°  C.  In  order  to  thrive,  the  species  requires  a  temperature 
never  going  below  22°  C.  at  night  and  rising  regularly  above  25°  C. 
during  the  day.  The  females  only  are  dangerous  as  sources  of  infection. 
The  insect,  like  Anopheles,  has  the  peculiarity  of  feeding  chiefly  at  night. 

Experiments  done  by  Reed,  Carroll,  Agramonte,  and  Lazear,^  to 
ascertain  whether  the  power  of  infecting  was  hereditarily  transmissible 
from  the  mosquito  to  following  generations,  were  negative.  A  positive 
result,  however,  has  been  reported  by  Marchoux  and  Simond.*  This 
question  must  still  await  more  extensive  research. 

Immunity. — Natural  immunity  against  yellow  fever  was  formerly 
assumed  to  exist  in  the  negro  race.  More  recent  investigations  have 
not  borne  out  this  assumption.  The  negro  soldiers  of  the  American 
army  in  Cuba  were  afflicted  equally  with  the  white  troops.  The  rela- 
tive immunity  of  dark-skinned  races,  however,  is  explained  possibly 
by  the  fact  that  the  stegomyia  prefers  to  attack  light-colored  surfaces. 

A  single  attack  seems  to  protect  against  subsequent  infection 
throughout  life. 

Artificial  immunization  has,  so  far,  been  unsuccessful.  Relative 
immunity  was  produced,  however,  by  Marchoux,  Salimbeni,  and 
Simond,  by  injections  of  the  serum  of  convalescents,  serum  heated  to 
55°  C,  and  of  defibrinated  blood  preserved  for  eight  days  in  vessels 
sealed  with  vaseline. 

1  Marchoux  and  Simond,  Ann.  de  I'inst.  Pasteur,  1906. 

«  Otto,  in  Kolle  und  Wassermann,  "Handbuch,"  etc.,  11,  Erganzungsband. 

■Loc.  cit. 

*  Marchoux  and  ^imorwf,  Comptes  rend,  de  la  soc,  de  biol.,  59,  J  905. 


CHAPTER  LI 

MEASLES,    SCARLET   FEVER,    TYPHUS    FEVER,    AND    FOOT- 
AND-MOUTH  DISEASE 

MEASL£S 

The  causative  agent  of  measles  is  unknown  to  the  present  day, 
and  it  would  be  a  thankless  task  to  review  the  literature  of  the  many 
attempts  to  isolate  microorganisms  from  this  disease,  none  of  which 
has  resulted  in  throwing  any  light  on  the  etiology. 

Attempts  to  produce  the  disease  experimentally  have  frequently 
been  made,  the  earliest  recorded  being  those  of  Home  of  Edinburgh, 
published  in  1759.^  Home  took  blood  from  the  arms  of  patients  afflicted 
with  measles,  and  caught  it  upon  cotton,  and  inoculated  normal  in- 
dividuals by  placing  this  blood-stained  cotton  on  to  wounds  made  in 
the  arm.  Home  claimed  that  in  this  way  he  produced  measles  of  a 
modified  and  milder  type  in  fifteen  individuals.  Home's  results,  how- 
ever, while  at  first  accepted,  were  assailed  by  many  writers  and  it  is 
by  no  means  certain  that  the  disease  produced  by  him  was  really  measles. 

A  number  of  other  observers  after  Home  attempted  experimental 
inoculation  of  this  disease,  and  positive  results  were  reported  by  Stewart 
of  Rhode  Island  (1799),  Speranza  of  Mantua  (1822),  Katowa  of  Hungary 
(1842),  and  McGirr  of  Chicago  (1850). 

The  experiments  of  all  these  early  writers,  however,  are  unsatisfac- 
tory, owing  to  the  necessarily  unreliable  technique  of  their  methods. 

In  1905,  Hektoen^  succeeded  in  experimentally  producing  the  dis- 
ease in  two  medical  students  by  subcutaneous  injection  of  blood  taken 
from  measles  patients  at  the  height  of  the  disease  (fourth  day).  The 
experiments  were  carefully  carried  out  and  the  symptoms  in  the  sub- 
jects were  unquestionable.  They  demonstrated  beyond  doubt  that  the 
virus  of  the  disease  is  present  in  the  blood.  Attempts  at  cultivation 
carried  out  with  the  same  blood  were  entirely  negative.  It  was  also 
shown  by  Hektoen's  experiments  that  the  virus  of  measles  may  be  kept 
alive  for  at  least  twenty-four  hours  when  mixed  with  ascitic  broth. 

1  Home,  "Medical  Facts  and  Experiments,"  Edinburgh,  1759. 

2  Hektoen,  Jour.  Inf.  Dis.,  ii,  1905. 

675 


676  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

SCARLET   FEVER 

(Scarlatina) 

The  etiology  of  scarlet  fever,  like  that  of  measles,  is  still  obscure. 
Streptococci  have  been  found  with  striking  regularitj^  in  the  throats  of 
scarlet-fever  patients,  and  a  large  number  of  investigations  have  seemed 
to  furnish  evidence  for  the  etiological  relationship  of  these  microorgan- 
isms with  the  disease. »  According  to  von  Lingelsheim,  Crooke  as  early 
as  1885  demonstrated  the  presence  of  streptococci  in  the  cadavers  of 
scarlet-fever  victims.  Baginsky  and  Sommerfeld  ^  in  1900  examined  a 
number  of  scarlatina  cases  with  reference  especially  to  streptococcus 
infection,  and  reported  the  presence  of  streptococci  in  the  hearths 
blood  of  eight  patients  who  had  died  after  a  very  acute  and  short 
illness.  They  expressed  the  belief  that  the  acuteness  of  the  illness  and 
the  rapidity  of  death  in  these  cases  precluded  the  possibility  of  the 
streptococci  being  merely  secondary  invaders.  A  large  number  of 
other  observers  have  expressed  similar  opinions,  but  we  can  not,  as 
yet,  justly  conclude  that  streptococci  are  actually  the  etiological 
agents  in  this  disease. 

Class  ^  in  1899  described  a  diplococcus  which  he  cultivated  from  a 
large  number  of  scarlatina  patients  and  with  which  he  was  able  to  pro- 
duce exanthemata  and  acute  fever  in  pigs.  Subsequent  investigations 
seem  to  show  that  Class  was  really  working  with  a  streptococcus. 

Moser,^  working  in  Escherich's  clinic,  has  recently  reported  the  very 
favorable  influence  upon  the  course  of  scarlet  fever  of  polyvalent 
streptococcus  antisera.  This  is  not  really  very  strong  evidence  in  favor 
of  the  streptococcus  etiology  of  the  disease,  since  there  is,  of  course,  no 
doubt  that  streptococcus  infection  complicates  the  disease,  and  it  is 
to  be  expected  that  antistreptococcus  serum  should,  therefore,  benefit 
the  patient's  condition  by  combating  this  complication. 

Mallory  ^  in  1904  published  observations  on  four  scarlatina  cases  on 
which  he  bases  the  belief  that  scarlatina  is  caused  by  protozoa.  In 
the  skin,  between  the  epithelial  cells,  he  found  small  bodies  which  were 
easily  stained  with  methylene-blue  and  which  because  of  their  arrange- 

1  Baginsky  and  Sommerfeld,  Berl.  klin.  Woch.,  1900. 

a  Class,  Phila.  Med.  Jour.,  iii,  1899. 

8  Moser,  quoted  by  Escherich,  Wien.  klin.  Woch.,  xxiii,  1903. 

*  Mallory,  Jour.  Med.  Research,  x,  1904. 


TYPHUS  FEVER  677 

ment  and  form  he  interpreted  as  parasites  not  very  unlike  the  Plasmo- 
dium of  malaria.  Subsequent  investigations  of  Field  ^  and  others  have 
failed  to  substantiate  Mallory's  conclusions. 

TYPHUS  FEVER 

Typhus  fever  is  an  infectious  disease  which  is  characterized  by  an 
incubation  time  of  5  days  or  more,  high  temperature,  and  a  petechial 
rash.  It  has  been  characterized  as  pecuUarly  a  disease  of  filth  and 
epidemically  disappeared  in  most  of  the  civiUzed  countries,  al- 
though it  is  still  endemic  in  certain  parts  of  Europe,  North  and  South 
America,  and  occurs  epidemically  in  Mexico  under  the  name  of  Tabar- 
dillo.  In  New  York  it  has  recently  been  found  to  exist  not  infrequently. 
It  was  described  as  a  new  clinical  entity  by  Brill,  and  has  been  spoken 
of  as  Brill's  disease,  but  the  work  of  Anderson  and  Goldberger  has 
shown  that  Brill's  disease  is  identical  with  typhus  fever.  Great  advances 
have  been  made  in  the  knowledge  of  the  disease  during  the  last  few  years. 

In  1909,  Nicolle^  successfully  inoculated  an  anthropoid  ape,  and 
Anderson  and  Goldberger  ^  in  the  same  year  succeeded  in  inoculating 
lower  monkeys,  rhesus  and  capuchin.  Similar  successful  monkey  in- 
oculations were  made  by  Ricketts  and  Wilder,^  by  Gavino  and  Girard.^ 
In  these  animals  inoculation  with  blood  from  active  cases  is  followed 
by  a  rapid  rise  of  temperature  after  an  incubation  time  of  5  days  or 
more,  and  the  fever  remains  high  for  3  to  5  days,  after  which  it  comes 
down  by  lysis.  Occasional  recrudescences  have  been  noticed  in  monkeys. 
Goldberger  and  Anderson  have  had  a  mortality  of  2  per  cent  in  their 
monkeys.  The  disease  may  be  transmitted  from  monkey  to  monkey 
with  the  blood,  which  is  infectious  during  the  febrile  period  and  may  be 
so  for  as  long  as  32  hours  after  the  temperature  returns  to  normal. 

The  disease  was  at  first  suspected  to  be  caused  by  a  filterable  virus, 
an  opinion  which  is  still  held  by  some  observers.  Most  workers  agree 
today,  especially  because  of  the  work  of  Anderson  and  Goldberger, 
that  filtered  blood  will  not  convey  the  disease,  and,  although  Nicolle, 
Conor  and  Conseil,  Ricketts  and  Wilder,  and  others  have  reported 

1  Field,  Jour.  Exper.  Med.,  vii,  1905.  ^  JSlicolle,  Compt.  rend.  Acad.  d.  Sc,  1909, 
p.  157;  Ann.  de  I'inst.  Past.,  1910,  1911,  1912. 

^Anderson  and  Goldberger,  Jour.  A.  M.  A.,  1912,  p.  49;  Jour.  Med.  Res.,  1910, 
p.  469;  N.  Y.  Med.  Jour.,  1912,  p.  976. 

4  Ricketts  and  Wilder,  Jour.  A.  M.  A.,  Feb.,  1910,  p.  463;  ibid.,  April  16,  1910, 
p.  1304;  ibid.,  April  23,  1910,  p.  1373;  ibid.,  July  23,  1910,  p.  309. 

^  Gavino  and  Girard,  cited  from  Anderson  and  Goldberger. 


678  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

that  occasionally  inoculation  with  filtered  blood  renders  monkeys  re- 
fractory to  later  inoculation,  it  is  generally  believed  at  present  that  the 
disease  is  caused  by  some  agent  too  large  to  pass  through  the  Berke- 
feld  or  Chamberland  filters. 

Work  on  the  etiology  of  typhus  has  been  very  extensive  and  many 
microorganisms  have  been  described. 

Ricketts  and  Wilder  saw  short  bacilli  in  smear  preparations,  but  were  not 
able  to  cultivate  them.  Rabinovitch  ^  described  a  Gram-positive  diplo-bacillus, 
cultivated  from  cases  of  an  epidemic  in  Kieff,  and  with  antigens  prepared  from 
this  organism,  he  obtained  complement-fixation  and  agglutination.  Fiirth 
studied  an  epidemic  in  China  and  obtained  short,  plump  rods  which  grew  aero- 
bically  in  short  chains.  P.  Th.  MuUer  saw  a  diplo-bacillus  upon  which  he  did 
not  lay  much  stress  etiologically,  and  Prowezek  described  inclusions  in  euco- 
cytes  which  he  regarded  as  protozoa.  It  is  hardly  worth  while  at  the  present 
time  to  describe  in  detail  the  many  different  findings  that  have  been  reported, 
since  in  few  of  them  is  there  sufficient  evidence  to  enable  us  to  come  to  con- 
clusions. 

In  1914  Plotz  2  described  a  short  Gram-positive  bacillus  which  he 
obtained  by  anaerobic  cultivation,  with  considerable  regularity,  from 
cases  of  Brill's  disease  at  the  Mt.  Sinai  Hospital,  New  York,  and  which 
since  then  has  been  made  the  subject  of  considerable  study  by  Plotz, 
Olitsky,  and  Baehr.^  They  have  obtained  the  bacillus  again  and  again, 
have  succeeded  in  obtaining  positive  agglutinations  and  complement- 
fixation  in  the  blood  of  endemic  typhus  cases  after  the  crisis  and  have 
obtained  a  similar  bacillus  from  a  number  of  European  typhus  cases 
which  have  come  into  quarantine. 

The  method  of  cultivation  by  which  this  bacillus  is  grown  is 
relatively  simple,  consisting  of  taking  blood  directly  from  a  vein  into 
high  tubes  containing  glucose  agar  and  unheated  and  unfiltered  ascitic 
fluid  of  a  specific  gravity  not  less  than  10.15. 

The  American  Red  Cross  Commission  which  went  to  Serbia  during  the  last 
typhus  epidemic — and  of  which  the  writer  was  a  member — attempted  to  work 
along  the  fines  laid  down  by  Plotz  but  found  it  extremely  difficult  to  do  sys- 
tematic work  and  obtain  reliable  materials  under  the  conditions  then  existing. 
The  undersigned  obtained  an  organism  very  similar  to  the  Plotz  bacillus  by 
Plotz's  method  in  two  cases.  In  the  first  of  these  the  organism  could  not  be 
carried  further  than  the  second  generation,  and  in  the  second  it  did  not  reach 
America  alive.    Hopkins  obtained  a  similar  organism  later  toward  the  end  of 

■  1  Rabinovitch,  Centralbk.  Bakt.  Orig.,  1909,  lii,  Arch.  f.  Hyg.,  1909. 
2  Plotz,  Jour,  of  A.  M.  A.,  Ixii,  20,  p.  14. 
'  Plotz,  Olitsky  and  Baehr,  Jour,  of  Inf.  Dis.,  xvii,  1915,  p.  1. 


TYPHUS  FEVER  679 

the  epidemic,  which  is  still  under  cultivation.  However,  these  organisms  were 
found  so  rarely,  in  spite  of  a  considerable  number  of  blood  cultures  that  were 
taken,  and  the  necessity  of  working  with  unsterihzed  ascitic  fluid  under  rela- 
tively primitive  conditions,  forced  the  conclusion  upon  them  that  these  isolated 
findings,  though  pointing  somewhat  in  favor  of  Plotz's  organism,  did  not  es- 
tabUsh  definite  proof.  However,  of  the  many  blood  cultures  taken,  the  smaller 
nimiber  were  taken  from  fresh  cases,  most  of  the  ascitic  fluid  used  did  not 
come  up  to  the  specifications  of  Plotz,  and  there  is  no  doubt  about  the  fact 
that  the  organism  described  by  Plotz  is  not  one  that  is  very  easily  cultivated 
unless  rich  ascitic  fluid  is  available. 

The  fact  that  the  Plotz  organism  has  been  cultivated  from  guinea- 
pigs  and  monkeys  experimentally  inoculated  is  further  evidence  in  favor 
of  this  bacillus. 

Petruschky  ^  has  recently  cultivated  a  similar  but  aerobic  bacillus  from 
sputum  in  typhus  cases  and  Arnheim  2  has  aerobically  cultivated  an  organism 
which  in  appearance  and  staining  properties  is  not  unhke  the  Plotz  bacillus. 
Amlieim  obtained  his  organism  from  six  cases,  on  ascitic  agar  plates,  on  which 
on  the  first  cultures  there  appeared  a  growth  hardly  visible  to  the  naked  eye 
and  which  in  transplants  continued  to  grow  very  delicately.  He  states  his 
organism  is  not  unUke  that  of  Petruschky  and  he  obtained  it  out  of  the  blood, 
the  sputum  and  the  urine  of  typhus  cases. 

There  appears  at  the  present  writing  to  be  much  evidence  in  favor 
of  the  bacillus  of  Plotz  as  the  causative  agent,  the  only  objection  to  its 
final  acceptance  being  that,  despite  the  fact  that  a  great  many  workers 
have  been  studying  this  disease  during  the  last  few  years,  the  micro- 
organisms which  have  been  described  have  not  been  similar  one  with 
the  other,  and  the  fact  that  the  Plotz  organism  seems  to  lose  its  viru:- 
lence  almost  immediately  upon  artificial  cultivation.  Also  according 
to  Anderson,  active  immunization  with  the  Plotz  bacillus  does  not 
render  guinea-pigs  refractory  to  virus  inoculation.  All  these  things  breed 
conservatism.  However,  with  a  relatively  simple  method  and  sufl&cient 
typhus  material,  the  question  of  the  Plotz  bacillus  should  soon  be  defi- 
nitely settled. 

The  transmission  of  typhus,  as  shown  by  Nicolle,  Ricketts  and 
Wilder,  and  Anderson  and  Goldberger  seems  to  be  mainly  through  the 
agency  of  the  louse, — the  body  louse  (pediculus  vestimenti)  pretty 
surely,  the  head  louse  (pediculus  capitis)  possibly,  the  pubic  louse 
probably  not.    So  far,  no  evidence  has  been  obtained  that  the  disease 

1  Petruschky,  Centralbk.  f .  Bakt.,  Ixxv,  1915,  p.  497. 

2  Arnheim,  D.  Med.  Woeh.,  36,  1916,  p.  1060. 


680  DISEASES  CAUSED  BY  FILTRABLE  VIRUS 

has  been  transmitted  by  the  flea  or  the  bedbug.  The  disease  is,  there- 
fore, one  particularly  likely  to  accompany  filthy  conditions  of  living, 
and  war,  and  famine.  The  louse,  when  obtaining  access  to  the  human 
body,  lives  largely  in  the  underclothing,  especially  along  the  seams, 
the  belt,  and  other  small  shelters,  feeding  two  or  three  times  a  day. 
The  sanitation  of  typhus  is,  therefore,  largely  a  campaign  against  lice, 
which  would  consist  in  attention  to  bedding,  sterilization  of  clothing, 
and  bathing.  Since  the  louse  is  very  susceptible  to  heat,  autoclave  or 
steam  sterilization  is  best  for  materials  that  can  stand  it, — clothing, 
etc.;  sulphur  properly  appHed  to  barracks,  stables,  etc.,  is  very  efficient; 
kerosene  or  gasolene  as  a  spray  for  the  hair  and  beard  is  excellent  as  a 
louse-killing  agent;  and,  for  rapid  louse-killing  in  underclothing,  it  has 
been  found  very  useful  to  drop  the  clothing  into  a  clothes  box  or  bag, 
with  about  an  ounce  of  chloroform,  for  several  hours. 

Most  authorities  believe  that  typhus  fever  is  transmitted  only  by 
vermin.  However,  during  the  recent  epidemic  in  Serbia,  it  was  the 
opinion  of  some  physicians  that  at  the  height  of  the  disease,  the  sputum 
eould  convey  it  by  infection.  This  is  very  uncertain.  By  the  work  of 
Nicolle  and  his  associates,  and  of  Ricketts  and  Wilder,^  also  of  Ander- 
son and  Goldberger,^  it  has  been  shown  that  the  virus  can  be  trans- 
mitted from  human  being  to  human  being  by  the  bites  of  the  body 
louse  (pediculus  vestimenti) ;  the  flea  and  the  bedbug  apparently  do  not 
transmit  the  disease.  The  head  louse  (pediculus  capitis)  may  possibly 
transmit  it. 

FOOT-AND-MOUTH  DISEASE 

This  malady  occurs  chiefly  in  cattle,  sheep,  and  goats,  more  rarely 
in  other  domestic  animals.  It  is  characterized  by  the  appearance  of  a 
vesicular  eruption  localized  upon  the  mucosa  of  the  mouth  and  upon  the 
delicate  skin  between  the  hoofs.  In  the  females  similar  eruptions  may 
appear  upon  the  udders.  With  the  onset  of  the  eruption  there  may  be 
increased  temperature,  refusal  of  food,  and  general  depression.  Usually 
the  disease  is  mild;  the  vesicles  become  small  ulcers  and  pustules.  Oc- 
casionally the  disease  is  complicated  by  catarrhal  gastroenteritis  or  an 
inflammation  of  the  respiratory  tract,  and  death  may  ensue. 

The  disease  is  transmitted  from  animal  to  animal  by  means  of  virus 
contained  in  the  vesicular  contents.  Infection  may  also  take  place 
through  the  agency  of  milk.     It  has  been  claimed,  moreover,  though 

^  Wilder,  Jour,  Inf.  Dis.,  July,  1911,  p.  9.  ^  Goldberger  and  Anderson,  Pub. 
Health  Report,  Wash.,   March,   1912;  ibid.,   May  31,  1912. 


FOOT-AND-MOUTH  DISEASE  681 

on  the  basis  of  insufficient  proof,  that  infection  may  take  place  through 
the  air,  without  actual  contact. 

Rarely  the  disease  may  be  transmitted  to  man.  Such  infection, 
when  it  does  take  place,  occurs  usually  among  the  milkers  and  attend- 
ants in  dairies,  and  is  transmitted  by  direct  contact.  The  disease  in 
man  is  usually  very  mild.  Mohler  states  that  the  disease  may  be  trans- 
mitted to  man  by  the  milk  of  infected  animals.  He  ^  adds  that  in  the 
United  States  the  disease  has  been  practically  eradicated. 

The  causative  agent  of  foot-and-mouth  disease  is  unknown.  A  num- 
ber of  organisms  have  been  cultivated  from  the  vesicles  and  mucous 
membranes  of  afflicted  animals,  but  none  of  these  could  be  shown  to 
have  etiological  significance.  Loeffler  and  Frosch,^  have  demonstrated 
that  the  virus  contained  in  the  vesicles  may  pass  through  the  pores  of  a 
filter.  The  virus  is  easily  destroyed  by  heating  to  60°  C.  and  by  com- 
plete desiccation. 

One  attack  of  foot-and-mouth  disease  protects  against  subsequent 
attacks.  This  immunity  in  most  cases  lasts  for  years,  though  rare  cases 
of  recurrence  within  a  single  year  have  been  reported.  Loeffler  has  ac- 
tively immunized  horses  and  cattle  with  graded  doses  of  virus  obtained 
from  vesicles  and  with  the  sera  of  such  animals  has  produced  passive 
immunity  in  normal  subjects. 

FILTRABLE  VIRUS 

Recent  investigations  into  the  causation  of  disease  have  revealed 
that  a  considerable  number  of  infections  may  be  caused  by  organisms 
too  small  to  be  held  back  by  filters  through  which  even  the  smallest 
bacteria  cannot  pass.  The  earliest  observations  of  such  ^'filtrable 
virus"  are  probably  those  of  Frosch  (1898)  in  foot-and-mouth  disease 
and  of  Beijerinck  in  the  mosaic  disease  of  tobacco.  Since  then  similar 
investigations  have  shown  that  a  large  nimiber  of  diseases  are  probably 
caused  by  such  minute  organisms;  their  investigation,  long  delayed 
by  the  belief  in  their  invisibility  by  even  the  most  powerful  microscopic 
aid,  and  by  our  inability  to  cultivate  them,  has  taken  new  impetus  from 
the  discovery  of  and  the  cultivation  of  minute  globoid  bodies  from  the 
virus  of  poliomyelitis  by  Flexner  and  Noguchi  (see  below).  The  follow- 
ing tabulation  is  based  largely  on  the  comprehensive  summary  published 
by  Wolbach.^ 

1  Mohler,  BuU.  No.  41,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash.,  1908. 

2  Loeffler  und  Frosch,  Cent,  f .  Bakt.,  1,  1908.  ' 

3  Wolbach.  Jour,  of  Med.  Res.,  xxvii,  1912. 


682 


DISEASES  CAUSED  BY  FILTRABLE  VIRUS 


Disease 

Transmission 

Occurrence 

Direct 

Indirect 

Yellow  fever 

Stegomyia  fasciata 

Man 

MoUuscum  contagiosum. 

Direct  contact 

Man 

Dengue  fever 

Culex  fatigans 

Man 

Verruca    vulgaris    filtra- 
bility 

? 

Man 

Trachoma?  filtrability. . . 

Direct 

Man 

Poliomyelitis 

Unknown;     proba- 
bly   nasal,    etc., 
discharges 

Indirect  by  stable- 
fly 

Man 

Measles  filtrability 

Direct 

Man 

claimed  by  Goldberger 
and  Anderson 

Scarlet  fever?  filtrability 

Probably  direct 

Man 

claimed  by  Cantacuzene 
and     Bernhardt     but 
doubtful 

Chimpanzee 

Foot-and-mouth  disease. 

Direct 

Man        rattle 

and  swine 

Rabies 

Direct  by  bite  with 
saliva 

Man    and    all 

naammals; 
birds 

Variola  and  vaccinia. , . . 

Direct 

Man,      cattle; 

transmitted 
to  monkeys, 
rabbits 

Pleuro-pneumonia  of 
cattle 

Direct 

Bovine  species 

African  horse-sickness. . . 

Probably      insects, 
mosquitoes 

Horses 

Sheep-pox 

Direct 

Sheep 

Cattle  plague 

Food  contaminated 
with  excreta 

Cattle 

Hog-cholera 

Direct 

Hogs 

Swamp  fever  of  horses .  . , 

Probably     indirect 
by  insects 

Horses 

Agalactia  of  sheep  and 
goats 

Contact 



Sheep  and 
goats 

"Blue  tongue" 

? 

? 

Sheep 

Guinea-pig  epizootic 

? 

? 

Guinea-pigs 

Guinea-pig  paralysis 

? 

? 

Guinea-pigs 

Novy's  rat  disease 

? 

? 

Rats 

Fowl  pest 

Feces 

Ph  easan  ts , 

sparrows, 
geese 

Fowl  diphtheria 

Contact    exudates, 
etc. 

Fowl 

Rous's  chicken  sarcoma. . 

? 

? 

Chickens 

Pappataci    (three-day 
fever) 

Unknown 

Phlebotomus  papa- 
tasii 

Man 

'      SECTION  V 

BACTERIA   IN    AIR,  SOIL,  WATER,  AND   MILK 


CHAPTER  LII 
BACTERIA   IN  THE   AIR  AND  SOIL 

BACTERIA   IN    THE   AIR 

Bacteria  reach  the  air  largely  from  the  earth's  surface,  borne 
aloft  by  currents  of  air  sweeping  over  dry  places.  Their  presence  in 
air,  therefore,  is  largely  dependent  upon  atmospheric  conditions ;  humid- 
ity and  a  lack  of  wind  decreasing  their  numbers,  dryness  and  high 
winds  increasing  them.  MultipUcation  of  bacteria  during  transit 
through  the  air  probably  does  not  take  place. 

Apart  from  these  considerations  the  presence  of  bacteria  in  air  also 
depends  upon  purely  local  conditions  prevailing  in  different  places. 
They  are  most  plentiful  in  densely  populated  areas  and  within  buildings, 
such  as  theaters,  meeting  halls,  and  other  places  where  large  numbers  of 
people  congregate.  On  mountain  tops,  in  deserts,  over  oceans,  and  in 
other  uninhabited  regions,  the  air  is  comparatively  free  from  bacteria. 
A  classical  illustration  of  this  fact  is  found  in  the  experiments  which 
Pasteur  carried  out  in  his  refutation  of  the  doctrine  of  spontaneous 
generation.  Tyndall  also,  in  working  upon  the  same  subject,  demon- 
strated this  fact.  From  the  surface  of  the  ground  and  other  places 
where  bacteria  have  been  deposited,  they  reach  the  air  only  after 
complete  drying.  It  is  a  fact  of  much  importance,  both  in  bacterio- 
logical work  and  in  surgery,  that  bacteria  do  not  rise  from  a  moist 
surface.  From  dry  surfaces  they  may  rise,  but  only  when  the  air  is 
agitated  either  by  wind  or  by  air-currents  produced  in  other  ways. 
In  closed  rooms,  therefore,  even  when  bacteria  are  plentiful  and  the  walls 
and  floors  are  perfectly  dry,  there  is  little  danger  of  the  inhalation  of 
bacteria  unless  the  air  is  agitated  in  some  way.  The  most  favorable 
conditions  for  the  occurrence  of  many  bacteria  in  air  are  the  existence 
of  a  prolonged  drought  followed  by  a  dry  wind.     Under  such  condi- 

683 


684  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

tions,  even  the  dark  places  and  unlighted  corners  of  streets  and  habita^ 
tions  are  thoroughly  dried  out,  and  bacteria  are  taken  up  and  carried 
about  together  with  particles  of  dust.  At  such  times  the  dangers  from 
inhalation  are  much  multiplied.  By  experimenjis  made  in  balloons, 
it  has  been  found  that  bacteria  are  plentiful  below  altitudes  of  about 
fifteen  hundred  feet  and  may  be  present,  though  much  reduced  in 
numbers,  as  high  up  as  a  mile  above  the  earth's  surface.  The  species 
of  bacteria  found  in  the  air  are,  of  course,  subject  to  great  variation, 
depending  upon  locality.  Molds  and  spore-forming  bacteria,  bein^ 
more  regularly  resistant  to  the  effects  of  sunlight  and  drying  thau 
bacteria  possessing  only  vegetative  forms,  are  naturally  more  generally 
distributed. 

Out  of  air  thus  laden  with  bacteria,  they  may  again  settle  when  the 
wind  subsides  and  the  air  becomes  quiescent.  The  process  of  settling, 
however,  is  extremely  slow,  since  the  weight  of  a  bacterium  is  probably 
less  than  a  billionth  of  a  gram,  and  it  may  be  held  in  suspension  in  air 
for  considerable  periods.  Rains,  snow,  or  even  the  condensation  of 
moisture  from  a  humid  atmosphere,  hastens  this  process  considerably, 
and  large  quantities  of  bacteria  may  settle  out  from  air,  in  a  com- 
paratively short  time,  in  ice  chests,  in  operating  rooms,  or  in  other  places 
in  which  much  condensation  of  water  vapor  takes  place. 

The  importance  of  the  air  as  a  means  of  conveying  disease  is  still  a 
problem  upon  which  much  elucidation  is  needed.  The  importance  of 
this  manner  of  conveyance  in  smallpox,  in  measles,  in  scarlet  fever, 
and  in  other  exanthemata,  can  not  be  denied.  As  regards  the  dis- 
eases of  known  bacterial  origin,  conveyance  by  air  is  of  importance 
in  the  case  of  tuberculosis,  where  infection  by  inhalation  may  take 
place,  and  in  the  case  of  anthrax,  where  inhaled  anthrax  spores  may 
give  rise  to  the  pulmonary  form  of  the  disease.  The  importance  of  air 
conveyance  for  any  great  distance  in  pneumonia,  in  influenza,  in  diph- 
theria, and  in  meningitis  is  by  no  means  clear  and  requires  much  fur- 
ther study.  The  expulsion  of  bacteria  from  the  lungs  and  naso-pharynx 
does  not  take  place  during  simple  expiration,  since  an  air-current  pass- 
ing over  a  moist  surface  is  not  sufficient  to  dislodge  microorganisms. 
Expulsion  of  bacteria  in  these  conditions  must  take  place  together  with 
small  particles  of  moisture  carried  out  in  sneezing,  coughing,  or  any 
forced  expiration.  The  bacteria  thus  discharged  are  then  subject  to  the 
process  of  drying  and  often  are  exposed  to  direct  sunlight  for  a  con- 
siderable period  before  they  are  again  taken  up  in  the  air. 

The  methods  of  estimating  the  bacterial  contents  of  the  air  are  not 


BACTERIA  IN  THE  AIR  AND  SOIL  685 

entirely  satisfactory.  The  simple  exposure  of  uncovered  gelatin  or  agar 
plates  for  a  definite  length  of  time,  and  subsequent  estimation  of  the 
colonies  upon  the  plates,  yield  a  result  which  is  variable  according  to  the 
air-currents  and  the  degree  of  moisture  in  the  atmosphere,  and  furnish 
no  volume  standard  for  comparative  results.  The  methods  which 
are  in  use  at  the  present  time  depend  upon  the  suction  of  a  definite 
quantity  of  air  by  means  of  a  vacuum-pump  through  some  substance 
which  will  catch  the  bacteria.  One  of  the  first  devices  used  for  this  pur- 
pose was  that  of  Hesse,  who  sucked  air  through  a  piece  of  glass  tubing, 
about  70  cm.  long  and  about  3.5  cm.  in  diameter,  the  inner  surface  of 
which  had  been  coated  with  gelatin  in  the  manner  of  an  EsmarchroU 
tube.  This  method  is  not  efficient,  since  a  large  number  of  the  bacteria 
may  pass  entirely  through  the  tube  without  settling  upon  the  gelatin. 
One  of  the  most  satisfactory  methods  at  present  in  use  is  that  in  which 
definite  volumes  of  air  are  sucked  through  a  sand-filter.  Within  a 
small  glass  tube,  a  layer  of  sterilized  quartz  sand,  about  4  cm.  in 
depth,  is  placed.  The  sand  is  kept  from  being  dislodged  by  a  small 
wire  screen.  After  the  air  has  been  sucked  through  the  filter  the 
sand  ts  washed  in  a  definite  volume  of  sterile  water  or  salt  solution, 
and  measured  fractions  of  this  are  planted  in  agar  or  gelatin  in  Petri 
plates.  The  colonies  which  develop  are  counted.  Thus,  if  two  liters 
of  air  have  been  sucked  through  the  filter,  and  the  sand  has  been 
washed  in  10  c.c.  of  salt 'solution,  and  1  c.c.  of  this  is  planted,  with 
the  result  of  fifteen  colonies,  then  the  two  liters  of  air  have  contained 
one  himdred  and  fifty  bacteria. 


BACTERIA   IN   SOIL 

Besides  the  normal  bacterial  inhabitants  of  the  soil,  bacteria  reach 
the  soil  from  the  air,  in  contaminated  waters,  in  the  dejecta,  excreta, 
and  dead  bodies  of  animals  and  human  beings,  and  in  the  substance  of 
decaying  plants.  It  is  self-evident,  therefore,  that  the  distribution  of 
bacteria  in  soil  depends  largely  upon  the  density  of  population  and  the 
use  of  the  soil  for  agricultural  or  other  purposes.  Thus,  bacteria  are  most 
plentiful  in  the  neighborhood  of  cess-pools  or  in  manured  fields  and  gar- 
dens. Such  conditions,  however,  may  be  regarded  as  abnormal.  Even 
in  uncultivated  fields  there  is  a  constant  bacterial  flora  in  the  soil  which 
is  of  great  importance  in  its  participation  in  the  nitrogen  cycle,  a  phase 
of  the  bacteriology  of  soil  which  has  been  discussed  in  detail  in  another 


686  BACTERIA  IN  AIR,' SOIL,  WATER,  AND  MILK 

section.  (See  page  40.)  There  are,thus,|regular  and  normal  inhabitants 
of  the  soil  which  fulfil  a  definite  function  and  may  be  found  wherever 
plant  life  flourishes.  In  addition  to  these^  innumei'able  varieties  of  sapro- 
phytes and  pathogenic  germs  may  be  present,  which  vary  in  species 
and  in  number  with  local  conditions.  Numenous  investigations  into 
the  actual  numerical  contents  of  the  soil  have  been  made.  Houston  ^ 
found  an  average  of  1,500,000  bacteria  per  gram  in  garden  soil,  and 
about  100,000  bacteria  per  gram  in  the  arid  soil  of  uncultivated  regions. 
Fraenkel,^  in  studying  the  horizontal  distribution  of  bacteria  in  the  earth, 
has  found  that  they  are  most  numerous  near  the  surface,  a  gradual 
diminution  occurring  down  to  a  depth  of  about  two  yards.  Beyond 
this,  the  soil  may  be  often  practically  sterile. 

Pathogenic  bacteria  may  at  times  be  found  in  the  surface  layers, 
and  these  are  often  of  the  spore-bearing  varieties.  Most  important 
among  them  from  the  medical  standpoint  are  the  bacillus  of  tetanus,  of 
malignant  edema,  and  the  Welch  bacillus.  If  a  guinea-pig  is  inoculated 
subcutaneously  with  an  emulsion  of  garden  soil,  death  will  result  almost 
invariably  with  enormous  bloating  and  swelling  of  the  body  due  to  gas 
production.  This  is  due  to  the  fact  that  the  spore-bearing,  gas-producing 
anaerobic  bacilli  are  commonly  present  and  are  actively  pathogenic  for 
these  animals.  The  frequent  occurrence  of  tetanus  in  persons  sustain- 
ing wounds  of  the  bare  feet  and  hands  in  fields  and  excavations,  is  a 
matter  of  common  knowledge.  Anthrax,  also,  may  be  easily  conveyed 
by  soil  in  localities  where  animals  are  suffering  from  this  infection.  It 
is  not  probable  that  pathogenic  germs  which  are  not  spore-bearers  sur- 
vive in  the  soil  for  any  great  length  of  time.  Unless  the  soil  is  specially 
prepared  by  the  presence  of  defecations  or  other  other  organic  material, 
the  nutrition  at  their  disposal  is  not  at  all  suitable  for  their  needs, 
since  rapid  decomposition  of  organic  materials  by  saprophytes  is  always 
going  on  in  the  upper  layers.  Furthermore,  in  the  deeper  layers  the  con- 
ditions of  temperature  and  possibly  oxygen  supply  are  not  at  all  favorable 
for  the  growth  of  most  pathogenic  bacteria.  Within  a  short  distance  from 
the  surface  the  temperature  of  the  soil  usually  sinks  below  14°  or  15°  C. 
An  interesting  series  of  experiments  by  Fraenkel  ^  have  demonstrated 
this  point.  This  investigator  buried  freshly  inoculated  agar  and  gelatin 
cultures  of  cholera  spirilla  and  of  typhoid  and  anthrax  bacilli  at  differ- 
ent levels,  and  examined  them  for  growth  after  two  weeks  had  elapsed. 

1  Houston,  Report  Med.  Officer,  Local  Govern.  Bd.,  London,  1897. 

^Fraenkel,  Zeit.  f.  Hyg.,  ii,  1887. 

8  Fraenkel,  Zeit.  f.  Hyg.,  xi,  1887.  . 


BACTERIA  IN  THE  AIR  AND  SOIL  687 

The  anthrax  bacilli  hardly  ever  showed  growth  at  a  depth  below  about 
two  yards,  and  cholera  and  typhoid  developed  colonies  at  these  depths 
only  during  the  summer  months.  Under  natural  conditions  it  must  be 
remembered  that,  at  these  levels,  suitable  nutritive  material  is  not 
found. 

A  consideration  of  practical  importance  in  this  connection  is  the 
possibility  of  infection  by  means  of  buried  cadavers.  An  elaborate  series 
of  experiments  has  been  carried  out  upon  this  subject  in  Germany,  with 
results  which  demonstrate  that  the  danger  from  the  burial  of  persons 
dead  of  infectious  diseases  was  formerly  much  exaggerated.  Experi- 
ments *  usually  failed  to  reveal  the  presence  of  cholera  and  typhoid 
bacilli  within  two  to  three  weeks  after  burial,  and  tubercle  bacilli  were 
never  found  after  three  months  had  elapsed.  It  was  only  in  the  case 
of  sporulating  microorganisms,  such  as  the  anthrax  bacillus,  that  the 
living  incitants  could  be  found  for  as  long  as  two  years  after  burial.  The 
dangers  of  infection  of  human  beings  through  the  agency  of  soil, 
therefore,  are  chiefly  those  arising  from  the  spore-bearing  bacteria  which 
are  able  to  remain  alive  in  spite  of  the  unfavorable  cultural  conditions. 
It  has  been  found  by  some  observers,^  however,  that,  under  special  con- 
ditions, non-sporulating  bacteria,  more  especially  the  typhoid  bacillus, 
may  remain  alive  in  soil  for  several  months.  Although  these  bacteria, 
as  well  as  those  of  cholera,  diphtheria,  etc.,  can  not  proliferate  under  the 
conditions  found  in  the  soil,  the  fact  that  they  can  remain  viable  for  such 
prolonged  periods  in  the  upper  layers  suggests  the  possibility  of  danger 
from  the  use  of  unwashed  vegetables,  such  as  lettuce  or  radishes  or  other 
soil  and  sewage  contaminated  food  products.  The  examination  of  soil 
for  colon  baciUi,  while  demonstrating  the  presence  or  absence  of  manure 
or  sewage  contamination,  has  no  practical  value,  since  colon  bacilli  are  * 
found  in  the  dejecta  of  animals. 

Examination  of  specimens  of  soil  for  their  numerical  bacterial 
contents  is  extremely  unsatisfactory  because  the  bacteria  there  found 
can  hardly  ever  all  be  cultivated  together  under  one  and  the  same 
cultural  environment.  A  large  number  are  anaerobic,  others  again 
thrive  at  low  temperatures,  while  again  another  class  may  require  un- 
usually high  temperatures.  When  such  examinations  are  made,  how- 
ever, specimens  of  the  soil  from  the  surface  layer  may  be  taken  in  a 
sterile  platinum  spoon.     When  taken  from  the  lower  levels,  a  drill, 

1  Petri,  Arb.  a.  d.  kais.  Gesundheitsamt,  vii. 

2  FiHh  and  Horrocks,  Brit.  Med.  Jour.,  Sept.,  1002. 
45 


688  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

such  as  that  devised  byFraenkel,  maybe  used.  This  consists  of  an  iron 
rod  the  lower  end  of  which  is  pointed.  Just  above  the  point  a  movable 
collar  is  fitted.  This  collar  has  a  slit-like  opening.  The  rod  beneath  the 
collar  has  a  deep  longitudinal  groove  corresponding  to  the  slit  in  the 
collar.  A  flange  on  the  collar  permits  opening  and  closing  of  the  groove 
while  the  instrument  is  below  the  ground.  The  drill  is  forced  into  the 
earth  to  the  desired  depth,  the  groove  is  opened  and  earth  is  forced  into 
the  chamber  by  twisting  the  rod.  In  the  same  manner  the  groove  may 
be  closed.  The  soil  obtained  in  this  way  is  taken  out  of  the  chamber 
and  a  definite  quantity,  say  one  gram,  is  dissolved  and  washed  thor- 
oughly in  a  measured  volume  of  sterile  water  or  sterile  salt  solution. 
Fractions  of  this  are  then  mixed  with  the  culture  medium,  plated,  and 
cultivated  aerobically  or  anaerobically  as  desired. 


CHAPTER  LIII 
BACTERIA  IN  WATER 

All  natural  waters  contain  a  more  or  less  abundant  bacterial  flora. 
This  fact,  combined  with  our  knowledge  that  the  incitants  of  several 
epidemic  diseases  and  a  number  of  minor  ailments  of  a  diarrheal  char- 
acter are  water  borne,  gives  the  bacteriological  investigation  of  water  a 
place  of  great  importance  in  hygiene.  In  nature,  there  are  very  few 
sources  of  water  supply  which  do  not  contain  bacteria  of  one  or  another 
description.  While  pathogenic  bacteria  are  usually  not  present  except 
in  those  waters  which  are  directly  contaminated  from  human  sources,  a 
thorough  understanding  of  the  quantitative  and  qualitative  bacterial 
contents  of  all  natural  waters  is  necessary  in  order  that  we  may  in- 
telligently gather  comparative  data  as  to  the  fitness  of  any  given  water 
for  human  consumption. 

The  gross  appearance  of  water  is  rarely,  if  ever,  an  indication  of  its 
danger.  The  turbid  waters  of  running  streams  in  sparsely  populated 
agricultural  districts  may  be  safe,  while  perfectly  clear  well  waters 
subjected  to  the  dangers  of  contamination  from  neighboring  sinks 
or  cess-pools  may  contain  large  numbers  of  pathogenic  germs. 

The  diseases  which  are  known  to  be  more  directly  connected  with 
water  supply  are  typhoid  fever  and  cholera. 

Typhoid  germs  discharged  from  the  bowel  or  from  the  urine  of 
typhoid  patients  or  convalescents  may  be  carried  by  the  sewage  or  from 
the  neighboring  soil  into  a  river  or  lake  and  lead  to  infection  of  the 
population  deriving  its.  drinking  water  from  this  source.  There  are  a 
great  many  investigations  on  record  in  which  severe  typhoid  epidemics 
have  been  traced  to  such  sources. 

In  the  case  of  cholera,  where  the  germs  are  discharged  from  the  bowels 
in  enormous  numbers,  conveyance  of  the  disease  by  water  is  even  more 
apparent,  and  the  discoverers  of  the  cholera  germ  themselves,  in  their 
early  work  in  Egypt  and  India,  were  able  to  isolate  the  bacteria  from 
contaminated  water  supplies. 

In  regard  to  the  less  clearly  understood  diarrheal  diseases,  dysen- 
tery, cholera  infantum,  etc.,  the  direct  relation  to  water  supply  has  not 

689 


690  BACTTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

been  so  definitely  proven,  and  can  be  deduced  only  from  the  diminu- 
tion of  such  infections  after  the  substitution  of  pure  water  for  the  pre- 
viously used  impure  supply.  It  is  thus  seen  that  water  bacteriology  is 
one  of  the  most  important  branches  of  the  science  of  hygiene,  and  has 
led,  and  is  constantly  leading,  to  enormous  diminution  of  the  death  rate 
in  all  communities  where  an  intelligent  study  of  the  conditions  has  been 
made. 

The  bacterial  purity  of  natural  waters,  although  dependent  upon 
special  and  local  conditions  in  relation  to  possible  contamination,  differs 
widely,  according  to  the  source  from  which  such  waters  are  derived. 

Rain  water  and  snow  water  are  usually  contaminated  with  bacteria 
by  the  dust  which  thej''  gather  on  their  way  to  the  ground,  and  are 
especially  rich  in  bacteria  when  taken  during  the  first  few  hours  of  a 
rain  or  snow  storm  when  the  air  is  still  dusty  and  filled  with  floating 
particles.  During  the  later  hours  of  prolonged  storms,  rain  water  and 
snow  water  may  be  comparatively  sterile.  Miquel,^  who  made  exten- 
sive experiments  in  France  on  the  bacterial  contents  of  rain  water, 
found  that  in  country  districts,  where  the  air  is  less  dusty,  rain  water 
contained  an  average  of  about  4.3  bacteria  to  the  cubic  centimeter. 

The  bacterial  counts  of  snow  water  are  usually  somewhat  higher  than 
those  of  rain. 

The  waters  of  streams,  ponds,  and  lakes  are  usually  spoken  of  as 
surface  waters,  and  these  of  all  natural  supplies  contain  the  largest  num- 
ber of  bacteria.  In  each  case,  of  course,  the  quantitative  and  quali- 
tative bacterial  flora  of  such  waters  is  intimately  dependent  upon  the 
conditions  of  the  surrounding  country,  the  density  of  the  population,, 
and  the  relation  of  these  waters  to  sewage.  It  is  also,  and  to  no  less; 
important  degree,  dependent  upon  weather  conditions,  the  influence  of 
light  and  temperature,  and  the  food  supply  contained  within  the  waters; 
in  the  form  of  decayed  vegetation.  In  all  such  surface  waters  there  is: 
constantly  going  on  a  process  of  self-purification.  The  chief  factor  in 
this  process  is  sedimentation.  In  stagnant  ponds  and  lakes  with  but- 
sluggish  currents  there  is  a  constant  sedimentation  of  the  heavier 
particles,  which  gradually  but  steadily  leads  to  a  diminution  of  the 
number  of  bacteria  in  the  upper  layers  of  the  water.  In  rivers  where 
sedimentation  is  to  a  certain  extent  prevented  by  rapidity  of  current, 
the  effectiveness  of  such  sedimentation  is,  of  course,  entirely  dependent 
upon  the  speed  of  the  current. 

^  Miguel,  Revue  dTiyg.,  viu,  1886. 


BACTERIA  IN  WATER  691 

The  influence  of  light  in  purifying  surface  waters  is  important  chiefly 
in  ponds,  lakes,  and  sheets  of  water  which  expose  a  large  surface  to  the 
sunlight,  and  where  the  surroundings  are  such  that  the  sun  has  free  access 
throughout  the  day.  According  to  the  researches  of  Buchner,^  the  bac- 
tericidal effects  of  light  penetrate  through  water  to  a  depth  of  three  feet. 

The  effects  of  temperature  in  purifying  surface  waters  under  natural 
conditions  are  probably  not  great.  There  is,  however,  a  general  tendency 
toward  diminution  of  the  bacterial  flora  as  the  temperature  of  such 
waters  becomes  lower. 

The  presence  of  protozoa  in  natural  waters  as  purifying  agents  has 
recently  been  emphasized  by  Huntemiiller,^  who  claims  that  these  organ- 
isms by  phagocytosis  greatly  diminish  the  number  of  bacteria  in  any 
given  body  of  water.  It  is  self-evident  that  the  number  of  bacteria  in 
any  of  these  waters  is  never  constant,  since  all  factors  which  tend  to  a 
diminution  or  increase  in  volume,  such  as  drying  up  of  tributary  streams 
or  the  occurrence  of  heavy  rains,  would  lead  to  differences  of  dilution 
which  would  materially  change  numerical  bacterial  estimations.  The 
influence  of  rains,  furthermore,  may  be  a  twofold  one.  On  the  one  hand, 
heavy  rain-falls,  by  washing  a  large  amount  of  dirt  into  the  rivers  and 
lakes  from  the  surrounding  land,  have  a  tendency  to  increase  the 
bacterial  flora.  This  influence  would  be  especially  marked  in  all  bodies 
of  water  which  are  surrounded  by  cultivated  land  where  manured  fields 
and  grazing-meadows  supply  a  plentiful  source  of  bacteria.  On  the 
other  hand,  in  regions  where  arid,  uninhabited  lands  surround  any 
given  river  or  lake,  the  rain  would  carry  with  it  very  little  living  con- 
tamination and  would  act  chiefly  as  a  diluent  and  diminish  the  actual 
proportion  of  bacteria  in  the  water. 

Another  and  extremely  important  source  of  water  supply  is  that 
spoken  of  technically  as  " ground  water."  The  ''ground  waters "  include 
the  shallow  wells  employed  in  the  country  districts,  springs,  and  deep 
or  artesian  wells.  The  shallow  wells  that  form  the  water  supply  for  a 
large  proportion  of  farms  in  the  eastern  United  States  are  usuaUy  very 
rich  in  bacteria  and  are  by  no  means  to  be  regarded  as  safe  sources,  ex- 
cept in  cases  where  great  care  is  observed  as  to  cleanliness  of  the  sur- 
roundings. In  such  wells  the  filtration  of  the  water  entering  the  well 
may  be  subject  to  great  variation  according  to  the  geological  con- 
ditions of  the  surrounding  ground.  The  proximity  of  barns  and  sinks 
may  lead  to  dangerous  contamination  of  such  waters. 

»  Btichner,  Arch.  f.  Hyg.,  xvii,  1895.       2  Huntemuller,  Arch.  f.  Hyg.,  liv  1905. 


692  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

Examinations  by  various  bacteriologists  have  shown  that  such  wells 
frequently  contain  as  many  as  five  hundred  bacteria  to  the  cubic  centi- 
meter. 

Perennial  spring  waters  are  usually  pure.  Examinations  by  the  Mas- 
sachusetts State  Board  of  Health  ^  in  1901  showed  an  average  of  about 
forty  bacteria  per  cubic  centimeter.  As  sources  of  water  supply  for 
general  consumption,  however,  springs  can  hardly  be  very  important 
because  of  the  insignificant  quantities  usually  derived  from  them. 

Of  much  greater  practical  importance  are  deep  artesian  wells,  which, 
under  ordinary  conditions,  are  largely  free  from  bacterial  contami- 
nation. 

Quantitative  Estimations  of  Bacteria. — The  quantitative  estima- 
tion of  bacteria  in  water  is  of  necessity  inexact,  because  of  the  difficulty 
of  always  securing  fair  average  samples  from  any  large  body  of  water, 
and  because  of  the  large  variations  in  cultural  requirements  of  the 
flora  present  in  them.  All  these  methods  depend  upon  colony  enumera- 
tion in  plates  of  agar  or  gelatin,  preferably  of  both.  For  the  sake  of 
gaining  some  basis  of  comparison  for  results  which,  at  best,  can  never  be 
entirely  accurate,  an  attempt  has  been  made  by  the  American  Public 
Health  Association  ^  to  standardize  the  methods  of  analysis. 

Water  for  analysis  should  always  be  collected  in  clean,  sterile  bottles, 
preferably  holding  more  than  100  c.c.  If  water  is  to  be  taken  from  a 
running  faucet  or  a  well  supplied  with  piping,  it  is  important  that  it 
should  be  allowed  to  run  for  some  time  before  the  sample  is  taken,  in 
order  that  any  change  in  bacterial  content  occurring  inside  of  the  pipes 
may  be  excluded.  It  is  obvious  that  in  water  pipes  through  which 
the  flow  is  not  constant,  bacteria  may  find  favorable  conditions  for 
growth  and  such  a  sample  would  not  represent  fairly  the  supply  to  be 
tested.  When  the  water  is  taken  from  a  pond,  lake,  or  cess-pool,  the 
bottle  may  be  lowered  into  the  water  by  means  of  a  weight,  or  may  be 
plunged  in  with  the  hand,  great  care  being  exercised  not  to  permit  con- 
tamination from  the  fingers  to  occur. 

After  the  water  has  been  collected  it  is  important  k)  plate  it  before 
the  bacteria  in  it  haye  a  chance  to  increase.  The  changes  taking  place 
during  transportation,  even  when  packing  in  ice  has  been  resorted  to, 
have  been  found  by  Jordan  and  Irons  ^  to  be  considerable.    It  is  impera- 

1  Mass.  State  Bd.  of  Health,  33d  Annual  Report  for  1901. 

2  Fuller,  Trans.  Amer.  Public  Health  Assn.,  xxvii,  1902.  Report  of  Com.  on 
Standard  Methods  of  Water  Analysis.    Jour.  Inf.  Dis.,  Suppl.  1,  1905. 

'  Jordan  and  Irons,  Reports  of  the  Amer.  Pub.  Hep-lth  Assn.,  xxv,  1889. 


BACTERIA  IN  WATER  693 

live,  therefore,  that  plating  of  the  water,  if  possible,  shall  not  be  delayed 
for  longer  than  one  or  two  hours  after  collection. 

Bacteriological  Examination  of  Water. — In  describing  the  meth- 
ods of  bacteriological  examinations  of  water,  we  adhere  strictly  to  the 
recommendations  of  the  Committee  on  Standard  Methods  of  the 
American  Public  Health  Association,^  taking  the  following  paragraphs 
with  slight  changes  from  their  report  of  1915: 

It  should  be  remembered  that  quantitative  estimations  of  bacteria 
in  water  are  of  most  value  when  repeatedly  done  and  a  ''normal"  for 
the  particular  water  supply  has  been  established,  so  that  deviation 
from  this  "normal"  can  be  easily  recognized.  Single  isolated  deter- 
minations may  easily  lead  to  error. 

The  following  paragraphs  are  taken  without  change  from  the  Public 
Health  Association's  report: 

"  Since  gelatine  does  not  give  the  total  number  of  bacteria  in  the  water,  the 
committee  has  thought  it  wise  to  use  agar  incubated  at  37°  C.  as  a  standard 
medium.  This  admits  of  counts  in  one  day  instead  of  two,  and  gives  results 
on  the  kind  of  bacteria  growing  at  blood  temperature  and  therefore  more 
nearly  related  to  pathogenic  types. 

"  Media. — The  standard  medium  for  determining  the  niunber  of  bacteria 
in  water  shall  be  nutrient  agar.  All  variations  from  this  medium  shall  be 
considered  special  media.  If  any  medium  other  than  standard  agar  is  used, 
this  fact  shall  be  stated  in  the  report. 

"For  general  work  the  standard  reaction  shall  be  -f-1  per  cent,  but  for  long 
continued  work  upon  water  from  the  same  source  the  optimum  reaction  shall 
be  ascertained  by  experiment  and  thereafter  adhered  to.  If  the  reaction  used, 
however,  is  different  from  the  standard,  it  shall  be  so  stated  in  the  report. 

"Procedure. — Shake  at  least  25  times  the  bottle  which  contains  the  sample. 
Withdraw  one  c.c.  of  the  sample  with  a  sterilized  pipette  and  deliver  it  into 
a  sterilized  Petri  dish,  10  cm.  in  diameter.  If  there  be  reason  to  suspect  that 
the  number  of  bacteria  is  more  than  200  per  c.c,  mix  one  c.c.  of  the  sample 
with  nine  c.c.  of  sterilized  tap  or  distilled  water.  Shake  25  times  and  measure 
one  c.c.  of  the  diluted  sample  into  a  Petri  dish.  If  a  higher  dilution  be  required 
proceed  in  the  same  manner,  e.g.,  ouq  c.c.  of  the  sample  to  99  c.c.  of  sterilized 
water,  or  one  c.c.  of  the  once  diluted  sample  to  nine  c.c.  of  sterilized  water,  and 
so  on.  In  the  case  of  an  unknown  water  or  a  sewage  it  is  advisable  to  use  sev- 
eral dilutions  for  the  same  sample".  To  the  liquid  in  the  Petri  dish  add  10  c.c.  of 
standard  agar  at  a  temperature  of  about  40°  C.  Mix  the  medium  and  water 
thoroughly  by  tipping  the  dish  back  and  forth,  and  spread  the  contents  uni- 
formly over  the  bottom  of  the  plate.    Allow  the  agar  to  cool  rapidly  on  a  hori- 

1  Amer.  P.  H.  A.,  Stand.  Meth.  Exam.  Water  and  Sewage,  1915. 


694 


BACTERIA  tN  AIR,  SOIL,  WATER,  AND  UlLK 


zontal  Surface  and  transfer  to  the  37°  C.  incubator  as  soon  as  it  is  hard. 
Incubate  the  culture  for  24  hours  at  a  temperature  of  37°  C.  in  a  dark,  well- 
ventilated  incubator  where  the  atmosphere  is  practically  saturated  with  mois- 
ture.V  After  the  period  of  incubation  place  the  Petri  dish  on  a  glass  plate 
suitably  ruled  and  count  the  colonies  with  the  aid  of  a  lens  which  magnifies 
at  least  five  diameters.  So  far  as  practicable  the  number  of  colonies  upon  the 
plate  shall  not  be  allowed  to  exceed  200.  The  wliole  number  of  colonies  upon 
the  plate  shall  be  Counted,  the  practice  of  counting  a  fractional  part  being 
resorted  to  only  in  case  of  necessity. 

"It  will  be  found  advantageous  to  use  Petri  dishes  with  porous  earthenware 
eovers  in  order  to  avoid  the  spreading  of  colonies  by  the  water  of  condensation.* 

"Expression  of  Results. — In  order  to  avoid  fictitious  accuracy  and  yet  to 
express  the  numerical  results  by  a  method  consistent  with  the  precision  of  the 
work  the  rules  given  below  shall  be  followed: 

"Numbers  of  Bacteria  per  c.c. 


From 

1  to               50  R 

ecorded 

as 

found 

(( 

51  "              100 

" 

to  the  nearest 

5 

it 

101  "              250 

(( 

(C 

(( 

10 

tt 

251  "              500 

(I 

tc 

It 

25 

u 

501  "           1,000 

(( 

(( 

u 

50 

tt 

1,001  "         10,000 

(( 

11 

t( 

100 

tt 

.10,001  "         50,000 

(( 

( 

It 

500 

tt 

50,001  "        100,000 

i( 

I 

(t 

1,000 

tt 

100,001  "       500,000 

(I 

t 

It 

10,000 

tt 

500,001  "     1,000,000 

I 

tt 

50,000 

(( 

1,000,001  "  10,000,000 

i 

It 

100,000" 

Qualitative  Bacterial  Analyses  of  Water. — Of  far  greater  iinportance* 
than  quantitative  analysis  is  the  isolation  of  bacteria  either  distinctly 
pathogenic,  such  as  the  cholera  spirillum  and  the  typhoid  bacillus, 
or  of  other  species  probably  emanating  from  contaminating  sources,, 
such  as  a  B.  coli.    Unfortunately  there  are  no  reliable  methods  by  which . 
typhoid  and  cholera  germs  can  be  isolated  from  water  with  auy  degree- 
of  regularity  or  certainty.     Although  frequently  the  isolation  of  such 
organisms  is  possible,  a  negative  result  in  these  cases  is  by  no  means 
eliminative  of  their  presence. 

The  isolation  of  typhoid  bacilli  from  water  is  very  difficult,  chiefly 
because  of  the  great  dilution  which  contaminations  undergo  upon  enter- 
ing any  large  body  of  water.  The  diflficulty  of  isolating  typhoid  bacilli,, 
even  from  the  stools  of  infected  patients,  makes  it  clear  that  such  diflft- 


1  Whipple,  Tech.  Quar.,  1899,  12,  p.  276. 
^Hill,  Jour.  Med.  Res.,  1904,  N.  S.,  8,  p.  93. 


BACTERIA  IN  WATER  695 

culties  are  enhanced  when  a  considerable  dilution  of  the  excreta  takes 
place.  Furthermore,  water  is  by  no  means  a  favorable  medium  for  the 
typhoid  bacillus.  Russell  and  Fuller  ^  have  shown  that  typhoid  bacilli 
may  die  in  water  within  five  days,  and  it  is  unquestionable  that  the 
rate  of  increase  of  these  bacteria  is  by  no  means  equal  to  that  of  many 
other  microorganisms  for  which  polluted  water  at  the  temperature  en- 
countered in  streams  and  lakes  forms  a  much  more  favorable  medium. 

It  is  thus  clear  that  even  in  infected  waters  the  number  of  typhoid 
bacilli  encountered  can  never  be  very  great.^ 

A  large  number  of  methods  for  the  isolation  of  the  typhoid  bacillus 
from  water  have  been  devised.  Most  of  the  media  used  are  identical 
with  those  employed  for  the  isolation  of  these  bacteria  from  the  stools. 
These  media  have  been  discussed  in  the  chapter  dealing  with  the  typhoid 
bacillus.  Success  is  rendered  more  likely  if  10  c.c.  of  the  water  is  first 
planted  into  lactose-bile  in  fermentation  tubes  holding  40  c.c.  After 
48  hours  at  37.5°  there  will  be  an  enrichment  of  typhoid  bacilli  which 
can  be  then  isolated  by  plating  in  the  usual  manner  (see  p.  134  and 
135)  on  Endo's  medium,  Conradi  Drigulski  or  any  of  the  other  usual 
differential  media. 

A  method  which  has  proved  useful  in  the  hands  of  Adami  and  Chapin  ^ 
is  one  which  depends  upon  the  phenomenon  of  agglutination.  They  at- 
tempt to  agglutinate  the  bacilli  out  of  liter  samples  of  water  by  adding 
powerful  agglutinating  serum. 

Vallet  and  others  have  attempted  to  precipitate  typhoid  bacilli  out 
of  water  by  chemical  means.  To  two  liters  of  water  add  20  c.c.  of  a  7.75 
per  cent  solution  of  sodium  hyposulphite  and  20  c.c.  of  a  10  per  cent  solu- 
tion of  lead  nitrate.  When  the  precipitate  has  settled,  the  clear  super- 
natant fluid  is  decanted  and  the  precipitate  dissolved  in  a  saturated 
sodium  hyposulphite  solution.  This  clear  solution  is  then  plated.  Will- 
son  ^  has  modified  this  method  by  adding  to  the  water  0.5  gm.  of  alum 
to  each  liter.  The  supernatant  fluid  is  removed  and  the  precipitate 
plated. 

The  isolation  of  the  vibrio  of  cholera  is  less  difficult  than  that  of  B. 
typhosus,  primarily  because  of  the  much  greater  numbers  of  these 
microorganisms  discharged  into  sewage.  The  number  of  cholera  spirilla 
in  the  excreta  of  cholera  patients  is  enormously  higher  than  is  that  of 

1  Russell  and  Fuller,  Jour.  Inf.  Dis.,  Suppl.  2,  1908. 

^  Laws  and  Anderson,  Rep.  of  Med.  Officer,  London  County  Council,  1894. 

3  Adami  and  Chapin,  Jour.  Med.  Res.,  xl,  1904, 

*  WiUs&n,  Jour,  of  Hyg.,  v,  1905, 


696  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

B.  typhosus  in  the  stools  of  typhoid-fever  patients.  It  is  not  infre- 
quent, therefore,  that  the  source  of  a  cholera  infection  may  be  directly 
traced  to  the  water  supply.  Koch,^  the  discoverer  of  the  cholera  vibrio, 
has  indicated  a  method  which  has  frequently  found  successful  application. 

To  100  c.c.  of  the  infected  water  are  added  one  per  cent  of  pepton 
and  one  per  cent  of  salt.  This  mixture  is  then  incubated  at  37.5°  C, 
and  after  ten,  fifteen,  and  twenty  hours,  specimens  from  the  upper 
layers  are  examined  microscopically  and  are  plated.  The  scum  from  the 
surface  of  such  a  medium  may  be  plated  on  the  starch  agar  of  Stokes 
and  Haechtel,^  on  which  colonies  of  intestinal  spirilla  will  appear  pink 
and  spreading. 

Because  of  the  great  difficulties  outlined  above  in  isolating  specific 
pathogenic  germs  from  polluted  waters,  bacteriologists  have  attempted 
to  form  an  approximate  estimation  of  pollution  by  the  detection  of  other 
microorganisms  which  form  the  predominating  flora  of  sewage.  Chief 
among  these  is  B.  coli.  The  isolation  and  numerical  estimation  of  B. 
coli  in  polluted  water  has  been  for  a  long  time  one  of  the  criteria  of  pollu- 
tion. This  so-called  colon  test,  however,  should  always  be  approached 
with  conservatism  and  should  never  be  carried  out  qualitatively  only. 
Careful  quantitative  estimation  should  be  made  in  every  case. 

B.  coli  in  water  is  by  no  means  always  the  result  of  human  con- 
tamination, since  this  bacillus  is  found  in  great  abundance  in  the  in- 
testines of  domestic  animals.  According  to  Poujol,  B.  coli  does  not 
even  always  point  to  fecal  contamination,  since  this  author  was  able  to 
find  the  bacillus  in  the  water  of  a  number  of  wells  where  no  possible 
contamination  of  any  sort  could  be  traced.  Prescott  ^  explains  this,  as 
well  as  similar  cases,  by  the  fact  that  organisms  of  the  colon  group  may 
occasionally  be  parasitic  upon  plants. 

The  opinions  of  hygienists  are  widely  at  variance  as  to  the  value  of 
the  colon  test.  While  the  discovery  of  isolated  bacilli  of  the  colon  group 
may  therefore  be  of  little  value,  it  is  nevertheless  safe  to  follow  the  opin- 
ion of  Houston,^  who  states  that  the  discovery  of  B.  coli  in  considerable 
numbers  invariably  points  to  sewage  pollution,  and  that  the  absolute 
absence  of  B.  coli  is,  as  a  rule,  reliable  evidence  of  purity. 

1  Koch,  Zeit.  f .  Hyg.,  xiv,  1893. 

2  Stokes  and  Haechtel,  see  Report  1915  A.  P.  H.  A.,  on  Water  Analysis.  The 
medium  is  an  agar  with  5.5  grams  agar,  5.0  meat  extract,  10.  Pepton  and  8.5  NaCl 
to  litre  to  which  is  added  10  grams  of  soluble  starch. 

3  Prescott,  Science,  xv,  1903. 

*  HoiLston,  Rep.  Medical  Officer,  Local  Gov.  Board,  London,  1900. 


BACTERIA  IN  WATER  697 

Rosenau  states  that  a  ground  water  should  be  condemned  even  if 
only  a  few  colon  bacilli  are  found,  for,  as  he  puts  it,  "these  bacteria  have 
no  business  in  a  soil-filtered  and  properly  protected  well  or  spring- 
water."  Surface  waters,  however,  may  easily  contain  a  few  colon  bacilli 
without  necessarily  having  been  exposed  to  contamination  by  human 
forces.  The  limit  of  safety,  Rosenau  states,  is  one  colon  bacillus  per  c.c. 
If  more  are  present  the  water  should  be  regarded  as  suspicious.  If  more 
than  10  per  c.c.  are  found  the  water  must  be  regarded  as  dangerous 
and  unqualifiedly  condemned. 

Presumptive  Colon  Tests. — For  this  purpose,  a  large  number  of 
methods  have  been  devised.  In  examining  sewage  or  other  polluted 
waters  in  which  the  number  of  colon  bacilli  is  comparatively  large,  the 
direct  use  of  lactose  litmus  agar  plates  jdelds  excellent  results.  The 
method  advised  by  the  American  Public  Health  Association  is  as  follows : 

"Add  the  quantities  of  water  or  sewage  to  be  tested  to  fermentation  tubes 
holding  at  least  40  cubic  centimeters  of  lactose  bile,^  incubate  at  37°  C.  and 
note  the  production  of  gas.  The  standard  time  for  observing  gas  production 
is  48  hours.  Small  numbers  of  somewhat  attenuated  B.  coli  may  require  three 
days  to  form  gas.  Attenuated  B.  coli  does  not  represent  recent  contamination 
and  all  B.  coli  not  attenuated  grows  readily  in  lactose  bile.  No  other  organism 
except  B.  Welchii  gives  such  a  test  in  lactose  bile.  B.  Welchii  is  of  rather  rare 
occurrence  in  water,  is  of  fecal  origin,  is  almost  invariably  accompanied  by  B. 
coli,  and  the  sanitary  significance  is  the  same. 

"A  comparison  of  the  positive  results  obtained  in  the  various  dilutions  of 
the  water  or  sewage  planted  into  the  lactose  bile  gives  a  good  idea  of  the  rela- 
tive amount  of  contamination  in  the  various  samples  examined. 

"  Qiiantities  of  Water  Tested. — For  ordinary  waters,  0.1, 1.0  and  10.0  c.c.  shall  be 
used  for  the  colon  test.  For  sewage  and  highly  polluted  surface  waters,  smaller 
quantities  shall  be  used;  and  for  ground  waters,  filtered  waters,  etc.,  the  quan- 
tities shall  be  larger,  if  necessary  to  obtain  positive  results.  The  quantities 
shall  vary  preferably  in  the  tenfold  manner  indicated.  Single  tests  with  quan- 
tities which  give  ordinarily  a  positive  result  or  ordinarily  a  negative  result  are 
in  themselves  of  but  little  account  for  quantitative  determinations.  The  range 
in  quantities  studied  shaU  be  sufficient  to  allow  the  quantities  needed  for  both 
a  positive  and  a  negative  result  to  be  recorded  for  each  sample.  When  this  is 
done,  the  results  of  several  tests  allow  an  approximate  estimate  of  the  number 
of  B.  coli  per  c.c." 

The  identification  of  colon  bacilli  so  obtained  should  then  be  under- 
taken. The  following  table,  again  taken  from  the  report  of  the  A.  P.  H. 
A.,  will  be  of  help: 

^  Prescott,  Science,  xvi,  1902. 


698 


BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 


O 

o 

o 
o 


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m  pq 


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M 


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+  +  I   +  I 

03 

§.        .        .        . 

^ 

P^ 

+    +    +      I  I 


03 


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+  +  +  +   +     II 


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+  I  +  + 

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03 

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+  +   I    +   I 

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+ 


CHAPTER  LIV 

BACTERIA     IN    MILK    AND    MILK    PRODUCTS,    BACTERIA     IN    THE 

INDUSTRIES 

BACTERIA    IN    MILE 

The  universal  use  of  cows'  milk  as  a  food,  especially  for  the  nourish- 
ment of  infants,  has  necessitated  its  close  study  by  bacteriologists  and 
hygienists.  It  furnishes  an  excellent  culture  medium  for  bacteria  and 
is,  therefore,  pre-eminently  fitted  to  convey  the  germs  of  infectious  dis- 
ease. The  many  changes  which  take  place  in  milk,  furthermore,  and 
which  add  or  detract  from  its  nutritive  value,  are  due  largely  to  bacterial 
growth  and  have  been  elucidated  b^  bacteriological  methods. 

Within  the  udder  of  the  healthy  cow,  milk  is  sterile.  If  pyogenic  or 
systemic  diseases  of  bacterial  origin  exist  in  the  cow,  the  milk  may, 
under  certain  circumstances,  be  infected  even  within  the  mammary 
glands.  In  the  milk  ducts  and  in  the  teats,  however,  even  in  perfectly 
healthy  animals,  a  certain  number  of  bacteria  may  be  found.  For  this 
reason,  even  when  all  precautionary  measures  are  followed,  the  milk 
as  received  in  the  pail  is  usually  contaminated.  As  a  matter  of  fact, 
the  anatomical  location  of  the  udder  and  the  mechanical  difficulties  of 
milking  make  it  practically  impossible  to  collect  milk  under  absolutely 
aseptic  conditions,  and,  under  the  best  circumstances,  from  100  to  500 
microorganisms  per  c.c.  may  usually  be  found  in  freshly  taken  milk. 
Withdrawn  under  conditions  of  ordinary  cleanliness,  the  bacterial 
contents  of  milk  are  considerably  higher  than  this.  After  the  proc- 
ess of  milking,  in  spite  of  all  practicable  precautions,  the  chances 
for  the  contamination  of  milk  are  considerable;  but  even  could  these 
be  eliminated,  the  bacterial  contents  of  a  given  sample  would  ultimately 
rapidly  increase  because  of  the  rich  culture  medium  which  the  milk 
provides  for  bacteria.  Whether  large  increases  shall  take  place  or  not  de- 
pends, in  the  first  place,  upon  the  temperature  at  which  milk  is  kept,  and, 
in  the  second  place,  upon  the  length  of  time  which  intervenes  before  its 
consumption.    Though  fresh  milk  possesses  slight  bactericidal  powers,^ 

1  Rosenau  and  McCoy,  Jour.  Med.  Res.,  18,  1908. 
699 


700  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

these  are  by  no  means  sufficient  to  be  of  practical  importance  in  the 
inhibition  of  bacterial  growth.  Kept  at  or  about  freezing-point,  the 
bacterial  contents  of  milk  do  not  appreciably  increase.  At  higher  tem- 
peratures, however,  a  rapid  propagation  of  bacteria  takes  place  which, 
especially  during  the  summer  months,  speedily  leads  to  enormous  num- 
bers. In  a  case  reported  by  Park,^  where  milk,  containing  at  the  first 
examination  30,000  microorganisms  per  cubic  centimeter,  was  kept 
at  30°  C.  (86°  F.)  for  twenty-four  hours,  the  count  at  the  end  of  this 
time  yielded  fourteen  billions  of  bacteria  for  the  same  quantity. 

It  is  of  much  importance,  therefore,  that  the  cleanliness  of  dairies, 
of  cattle,  and  in  the  handling  of  milk  should  be  reinforced  by  the  utmost 
care  in  chilling  and  icing  during  shipment  and  before  sale. 

Because  of  its  great  importance,  especially  for  the  health  of  the  chil- 
dren in  large  cities  during  the  summer  months,  the  milk  question  has, 
of  recent  years,  received  much  attention  from  health  officers.  In  the 
city  of  New  York,  the  question  has  been  made  the  subject  of  many 
careful  studies  by  Park  ^  and  his  associates.  Commissions,  working 
in  Chicago,^  Boston,*  and  other  large  towns,  have  placed  the  sale  of  milk 
under  more  or  less  exact  bacteriological  supervision.  Park  has  de- 
termined that  the  milk,  as  sold  in  New  York  stores  during  the  cold 
weather,  not  infrequently  averages  seven  hundred  and  fifty  thousand 
bacteria  per  cubic  centimeter;  during  the  hot  summer  months,  the 
bacterial  contents  of  similar  milk  not  infrequently  average  one  million 
and  more,  for  the  same  quantity.^  In  consequence  of  these  and  other 
researches,  large  dairies  have  introduced  bacteriological  precautions 
into  their  method  of  milk  production.  They  have  attempted  the  reduc- 
tion of  the  bacterial  contents  of  milk  by  scrupulous  cleanliness  of  the 
bams  and  of  the  udders  and  teats  of  the  cow,  by  the  elimination  of  dis- 
eased cattle,  by  sterilization  of  the  vessels  in  which  the  milk  is  received, 
and  of  the  hands  of  the  milker;  also  by  the  immediate  filtering  and 
cooling  of  the  milk. and  the  packing  of  the  milk  cans  in  ice,  where 
they  remain  until  delivered  to  the  consumer.  In  consequence  of  such 
measures,  it  is  possible  for  cities  to  be  supplied  with  milk  containing  no 
more,  and  often  less,  than  fifty  thousand  bacteria  to  the  cubic  centimeter. 
A  standard  of  cleanliness  has  been  established  in  various  towns  by  the 

1  Park,  W.  H.,  "Pathogenic  Bacteria,"  New  York,  1905,  p.  463. 

2  Park,  Jour,  of  Hygiene,  1,  1901. 

3  Jordan  and  Heinemann,  Rep.  of  the  Civic  Federation  of  Chicago,  1904« 
*  Sedgwick  and  Batchelder,  Bost.  Med.  and  Surg.  Jour.,  126,  1892, 

5  Escherich,  Fort.  d.  Medizin,  16  and  17,  1885, 


BACTERIA  IN  MILK  701 

introduction  of  the  so-called  "  certified  milk/'  which,  by  the  New  York 
Milk  Commission,  is  required  to  contain  no  more  than  thirty  thousand 
bacteria  per  cubic  centimeter.  Great  stress  is  laid  upon  such  numerical 
counts  simply  in  that  they  are  approximate  estimates  of  cleanliness. 
Most  of  the  bacteria,  however,  contained  in  milk  are  non-pathogenic, 
and  numbers  much  larger  than  the  maximum  required  for  certified  milk 
may  be  present  without  actual  disease  or  harm  following  its  consump- 
tion. 

The  various  species  of  bacteria  which  may  be  found  in  milk  include 
almost  all  known  varieties.  Whether  there  are  special,  so-called  milk 
bacteria  or  not  is  a  question  about  which  investigators  have  expressed 
widely  differing  opinions.  It  is  probable  that  many  of  the  species, 
formerly  regarded  as  specifically  belonging  to  milk,  are  there  simply  by 
virtue  of  their  habitual  presence  in  fodder,  straw,  or  bedding,  or  upon 
cattle.  It  is  likely,  furthermore,  that  some  of  these  species  are  found 
with  great  regularity  because  of  their  power  to  outgrow  other  species 
under  the  cultural  conditions  offered  them  in  milk. 

Under  normal  conditions,  milk  always  undergoes  a  process  which  is 
popularly  known  as  souring  and  curdling.  This  is  due  to  the  forma- 
tion of  lactic  acid  from  the  milk  sugar  and  is  the  result  of  the  enzymatic 
activities  of  several  varieties  of  bacteria  commonly  found  in  milk.  Most 
common  among  these  bacteria  is  the  so-called  Bacillus  lactis  aerogenes, 
an  encapsulated  bacillus  closely  related  to  the  colon-bacillus  group.  (See 
page  451.)  The  transformation  of  the  lactose  into  lactic  acid  may  occur 
either  directly  by  hydrolytic  cleavage: 

C„H^O„  +  H,0  =  4C3H„03; 

or  indirectly  through  a  monosaccharid, 

C„H,,0„  +  H3O  =  2C,H,3  0,  =  403H,03. 

Other  microorganisms  which  may  cause  lactic-acid  fermentation  in 
milk  are  the  so-called  Streptococcus  lacticus,  the  common  pyogenic 
streptococcus,  the  Staphylococcus  aureus.  Bacillus  coH  communis  and 
communior,  and  many  other  species.  Most  commonly  concerned  in  lactic- 
acid  production,  however,  according  to  Heinemann,^  are  the  two  first- 
mentioned,  Bacillus  lactis  aerogenes  and  Streptococcus  lacticus.  The 
secret  of  the  regularity  with  which  some  of  these  bacteria  are  present  in 
sour  milk  is  probably  found  in  the  ability  of  these  varieties  to  withstand 
a  much  higher  degree  of  acidity  of  the  culture  medium  than  other  species. 

>  Heinemann,  Jour,  of  Inf.  Dis.,  3,  1906. 


702  BACTERIA  IN  Attl,  ySOIL,  WATER,  AND  MiLiC 

In  consequence,  they  are  able  to  persist  and  develop  when  cultural  con. 
ditions  are  absolutely  unsuited  to  other  bacteria. 

Consequent  upon  acidification  of  the  milk  by  lactic-acid  formation, 
there  is  coagulation  of  casein.  Casein  precipitation,  however,  may 
also  be  due  to  a  non-acid  coagulation  caused  by  a  bacterial  ferment. 
Casein  precipitated  in  this  way  may  be  redissolved  by  a  bacterial 
trypsin  or  casease,  produced  by  the  same  or  other  bacteria,  the  milk 
again,  becoming  entirely  liquid,  transparent,  and  of  a  yellowish  color. 
The  casein  precipitated  by  lactic-acid  formation,  however,  is  never 
thus  redissolved,  because  the  high  acidity  does  not  permit  the  pro- 
teolytic ferments  to  act.^ 

Butyric-acid  fermentation  in  milk,  a  common  phenomenon,  is  also  an 
evidence  of  bacterial  growth.  As  a  rule,  it  is  produced  by  the  anaerobic 
bacteria,  and  is  a  process  developing  much  more  slowly  than  other  fer- 
mentations. A  large  number  of  bacteria  have  been  described  which 
are  capable  of  producing  such  changes,  the  chemical  process  by  which 
they  are  produced  being,  as  yet,  not  entirely  understood.  It  is  probable 
that  the  process  takes  place  after  hydrolysis  of  the  disaccharid  some- 
what according  to  the  following  formula: 

Ce  B.,2  Oe  =  C4  Hs  O2  +  2  CO2  +  2  H2. 

Special  bacteria  have  been  described  in  connection  with  this  form  of 
milk  fermentation,^  most  of  them  non-pathogenic.  It  is  unquestionable, 
however,  that  many  of  the  well-known  pathogenic  bacteria,  such  as 
Bacillus  aerogenes  capsulatus.  Bacillus  oedematis  •  maligni,  possess  the 
power  of  similar  butyric-acid  formation.  While  less  commonly  observed 
in  milk,  because  milk  is  rarely  kept  long  enough  to  permit  of  the  action 
or  development  of  these  enzymes,  the  butyric-acid  fermentation  is  of 
importance  in  connection  with  butter,  where  it  is  one  of  the  causes  pro- 
ducing rancidity. 

Alcoholic  fermentation  may  take  place  in  milk  as  a  result  of  the  ac- 
tivities of  certain  yeasts.  Upon  the  occurrence  of  such  fermentations 
depends  the  production  of  kefyr,  koumys,  and  other  beverages  which 
have  been  in  common  use  for  many  years,  especially  in  the  region  of  the 
Caucasus.  The  characteristic  quality  of  these  beverages  is  contrib- 
uted by  the  feeble  alcoholic  fermentation  produced  by  members  of  the 
saccharomyces  group,  but  side  by  side  with  this  process  lactio-acid  forma- 

»  Conn,  Exper.  Stat.  Rep.,  1892. 

a  Schattenfroh  uud  Grasberger,  Arch.  f.  Hyg.,  37,  1900. 


BACTERIA  IN  MILK  703 

tion  also  takes  place.  Beijerinck/  who  has  carefully  studied  the  so- 
called  kefyr  seeds,  used  for  the  production  of  kefyr  in  the  East,  has 
isolated  from  them  a  forrn  of  yeast  similar  in  many  respects  to  the 
ordinary  beer  yeast,  and  a  large  bacillus  to  which  he  attributes  the 
lactic-acid  formation. 

Occasional  but  uncommon  changes  which  occur  in  milk  lead  to  the 
formation  of  the  so-called  "  slimy  milk,"  yellow  and  green  milk,  and 
bitter  milk.  These  may  be  due  to  a  number  of  bacteria.  A  microorgan- 
ism which  is  commonly  found  in  connection  with  the  sHmy  changes  in 
milk  is  the  so-called  Bacillus  lactis  viscosus.  According  to  the  researches 
of  Ward,^  this  microorganism  is  frequently  derived  from  water  and  it  is 
the  water  supply  which  should  attract  attention  whenever  such  trouble 
occurs  in  dairies. 

The  so-called  blue,  green,  and  yellow  changes  are  usually  due  to 
chromogenic  bacteria,  such  as  Bacillus  cyanogenes.  Bacillus  prodigiosus, 
and  others. 

"Bitter  milk,"  a  condition  which  has  occasionally  been  observed  epi- 
demically, is  also  the  consequence  of  the  growth  of  microorganisms. 
Conn,^in  1891,  isolated  from  a  specimen  of  bitter  cream  a  diplococcus 
which  occasionally  forms  chains  and  which  in  sterilized  milk  develops 
rapidly,  producing  an  extremely  bitter  taste.  The  organism  of  Conn 
differs  from  a  similar  diplococcus  described  by  Wagmann  ^  in  that  it 
possesses  the  ability  of  producing  butyric  acid. 

Milk  in  Relation  to  Infectious  Disease. — As  a  source  of  direct  in- 
fection, milk  is  second  only  to  water,  and  deserves  close  hygienic  at- 
tention. A  large  number  of  infectious  diseases  have  been  traced  to  milk, 
although  the  actual  proof  of  the  etiological  part  played  by  it  in  such  cases 
has  often  been  difficult  to  adduce  and  has  necessarily  been  indirect. 
Nevertheless,  even  when  indirect  proof  only  has  been  brought,  it  has  been 
sufficiently  convincing  to  necessitate  the  most  careful  investigation  into 
milk  supplies  whenever  epidemics  of  certain  infectious  maladies  occur. 

Typhoid-fever  epidemics  have  been  frequently  traced  to  milk  in- 
fection, and,  in  this  disease,  milk  is,  next  to  water,  the  most  frequent 
etiological  factor.  Schiider,^  in  an  analysis  of  six  hundred  and  fifty 
typhoid  epidemics,  found  four  hundred  and  sixty-two  attributed  to 

>  Beijerinck,  Cent.  f.  Bakt.,  vi,  1889. 
2  Ward,  Bull.  165,  Cornell  Univ.  Agri.  Exp.  Stat.,  1899, 
»  C(mn,  Cent.  f.  Bakt.,  ix,  1891. 
»  Wagmann,  Milchztg.,  1890. 
*  Schuder,  Zeit.  f.  Hyg.,  xxxviii,  1901. 
46 


704         '         BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

water,  one  hundred  and  ten  to  milk,  and  seventy-eight  to  aii  other 
causes. 

Trask  ^  compiled  statistics  of  one  hundred"  and  seventy-nine  typhoid 
epidemics  supposed  to  have  been  caused  by  milk,  in  various  parts  of  the 
world.  In  all  such  epidemics  the  origin  of  infection  was  generally  trace- 
able to  diseased  or  convalescent  persons  employed  in  dairies,  to  con- 
taminated well  water  used  in  washing  milk  utensils,  or  to  the  use  of  cans 
and  bottles  returned  from  dwellings  where  typhoid  fever  had  existed. 
Actual  bacteriological  proof  of  the  infectiousness  of  milk  by  the  isolation 
of  Bacillus  typhosus  is  rare,  but  has  been  accomplished  in  isolated  in- 
stances. In  the  case  of  one  epidemic,  Conradi  ^  isolated  the  bacillus  from 
the  milk  on  sale  at  a  bakery  at  which  a  large  number  of  the  infected 
individuals  had  purchased  their  milk.  The  examination  of  market  milk 
at  Chicago,  through  a  period  of  eight  years,  revealed  the  presence  of 
typhoid  bacilli  but  three  times. 

In  spite  of  the  few  cases  in  which  actual  bacteriological  proof  has  been 
brought,  it  is  not  unlikely  that  careful  and  systematic  researches  would 
reveal  a  far  greater  number,  since  many  writers  have  shown  that  typhoid 
bacilli  may  remain  alive  in  raw  milk  for  as  long  as  thirty  day^,^  and 
may  actively  proliferate  in  the  milk  during  this  time.  One  peculiarity 
of  epidemics  which  may  aid  in  arousing  the  suspicion  that  they  have 
originated  in  milk  is  that,  in  such  cases,  women  and  children  are  far 
more  frequently  attacked  than  men.^ 

A  feature  which  adds  considerably  to  the  dangers  of  milk  infection 
is  the  unfortunate  absence  of  any  gross  changes,  such  as  coagulation, 
by  the  growth  of  typhoid  bacilli. 

Scarlet  fever,^  though  as  yet  of  unknown  etiology,  has  in  many  cases 
been  traced  indirectly  to  milk  infection.  Trask  has  collected  fifty-one 
epidemics  of  scarlet  fever  presumably  due  to,  milk.  In  one  epidemic 
occurring  in  Norwalk,  Conn.,®  twenty-nine  cases  were  distributed  among 
twenty-five  families  living  in  twenty-four  different  houses.  The  indi- 
viduals affected  did  not  attend  the  same  school,  and  were  of  entirely 
different  social  standing,  the  only  factor  common  to  all  of  them  being 
the  milk  supply. 

1  Trask,  Bull.  No.  41,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash. 

2  Conradi,  Cent.  f.  Bakt.,  I,  xl,  1905. 

»  Heim,  Arb,  a.  d.  kais.  Gesundheitsamt,  v. 

•  Wilckens,  Zeit.  f.  Hyg.,  xxvii,  1898. 

•  Trask,  loc.  cit. 

•  Herbert  E.  Smith,  Rep.  Conn.  State  Bd.  of  Health,  1897. 


BACTERIA  IN  MILK  705 

Diphtheria  has  been  frequently  traced  to  the  use  of  infected  milk. 
In  most  of  the  epidemics  reported  as  originating  in  this  way,  the  proof 
has  been  necessarily  indirect.  In  two  out  of  twenty-three  epidemics 
reported  by  Trask,  however,  Bacillus  diphtherise  was  isolated  from 
the  milk  directly.  The  ability  of  the  Klebs-Loeffler  bacillus  to  proliferate 
and  remain  aHve  for  a  long  while  in  raw  milk  has  been  demonstrated 
by  Eyre  ^  and  others. 

Whether  or  not  cholera  asiatica  may  be  transmitted  by  means  of 
milk  has  been  a  disputed  question.  Hesse  ^  claims  that  cholera  spirilla  die 
out  in  raw  milk  within  twelve  hours.  This  statement,  however,  has  not 
been  borne  out  by  other  observers.^  Unquestionable  cases  of  direct 
transmission  of  cholera  by  means  of  milk  have  been  reported  by  a  num- 
ber of  writers,  notably  by  Simpson."* 

The  relation  of  milk  to  the  diarrheal  diseases  of  infants  has,  of  late 
years,  received  a  great  deal  of  attention.  In  large  cities,  during  the 
summer  months,  numerous  cases  of  infantile  diarrhea  among  bottle- 
fed  babies  occur,  which,  in  many  instances,  are  attributed  to  feeding  with 
contaminated  milk.  Park  and  Holt,^  who  have  made  extensive  re- 
searches upon  this  question  in  New  York  City,  have  come  to  the  con- 
clusion that  the  harmful  effects  of  contaminated  milk  upon  babies 
can  not  be  ascribed  to  any  given  single  microorganism  in  the  milk. 
Specifically  toxic  properties  were  found  by  these  writers  for  none  of  the 
one  hundred  and  thirty-nine  different  species  of  bacteria  isolated  from 
unsterilized  milk.  It  is  unlikely,  therefore,  that  the  diarrheal  diseases 
among  babies  have  a  uniform  bacteriological  cause.  Whether  or  not 
these  diarrheal  conditions  depend  entirely  upon  the  bacterial  contents 
of  milk  or,  in  a  large  number  of  cases  at  least,  upon  the  inabiHty  of  the 
child  to  digest  cows'  milk  because  of  chemical  conditions,  must  be  left 
undecided.  Park  and  Holt,  in  analyzing  their  extensive  data,  conclude 
that  milk  containing  "  over  one  million  bacteria  to  the  cubic  centimeter 
is  certainly  harmful  to  the  average  infant." 

The  significance  of  the  presence  of  streptococci  in  milk,  as  an  element 
of  danger,  has  recently  received  much  attention  in  the  literature.  Heine- 
mann,®  who  has  made  a  careful  comparison  of   Streptococcus  lacticus 

'  Eyre,  Brit.  Med.  Jour.,  1899. 
^  Hesse,  Zeit.  f.  Hyg.,  xvii,  1894. 
\Basenau,  Arch.  f.  Hyg.,  xxiii,  1895. 

*  Simpson,  Indian  Med.  Gaz.,  1887. 

»  Park  and  Holt,  Arch,  of  Fed.,  Dec,  1903. 

*  Heinemann,  Jour.  Inf.  Dis.,  3,  1906. 


706  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

(formerly  spoken  of  as  Bacillus  acidi  lactici  [Kruse]),  with  other,  strep- 
tococci, has  shown  that,  essentially,  this  streptococcus  does  not  differ 
from  streptococci  from  other  sources,  and  is  practically  indistinguish- 
able by  cultural  methods  from  Streptococcus  pyogenes.  Similar  com- 
parisons made  by  Schottmiiller,^  Muller,^  and  others  have  led  to  like  re- 
sults. Since  streptococci  may  be  found  in  milk  from  perfectly  normal 
cows  and  are  almost  regularly  associated  with  lactic-acid  fermenta- 
tion, it  is  unlikely  that  these  microorganisms  hold  ordinarily  any 
specific  relationship  to  disease. 

Recently,  however,  a  number  of  epidemics  of  sore  throat  caused  by 
streptococci  have  been  traced  to  milk  upon  reasonably  reliable  evidence. 
Accounts  of  such  epidemics  in  Chicago  and  in  Baltimore  have  been 
published  by  Capps  and  Miller  ^  and  by  Hamburger.^ 

The  presence  of  pus  cells  and  leucocytes  in  milk,  together  with 
streptococci,  was  also  formerly  regarded  as  of  great  importance. 
Enumerations  of  leucocytes  in  milk  were  first  made  by  Stokes  and 
Weggefarth.^  Their  method  of  enumeration  consisted  in  centrifugaliz- 
ing  a  definite  volume  of  milk,  spreading  the  entire  sediment  over  a 
definite  area  on  a  slide,  and  counting  the  leucocytes  found  in  a  number  of 
fields.  Calculations  from  this  may  then  be  made  as  to  the  number  of 
leucocytes  per  cubic  centimeter.  This  method,  and  modifications  of  it, 
have  been  used  by  a  large  number  of  observers,  but  the  value  of  the  con- 
clusions drawn  from  them  has  been  much  exaggerated.  Normal  milk 
may  contain  leucocytes  in  moderate  numbers,  and  importance  may  be 
attached  to  such  leucocyte  counts  only  when  their  number  largely  ex- 
ceeds that  present  in  other  specimens  of  perfectly  normal  milk.  When- 
ever such  high  leucocyte  counts  are  found,  of  course,  a  careful  veteri- 
nary inspection  and  examination  for  pyogenic  disease  should  be  made. 

Foot-and-mouth  disease,  an  infectious  condition  prevailing  among 
cattle,  characterized  by  a  vesicular  rash  on  the  mouth  and  about  the 
hoofs,  has,  in  a  number  of  cases,  been  definitely  shown  to  be  transmitted 
to  man  through  the  agency  of  milk.  Notter  and  Firth^  reported  an 
epidemic  occurring  among  persons  supplied  with  milk  from  a  single  dairy 
in  which  foot-and-mouth  disease  prevailed  among  the  cows.     In  this 

1  Schottmuller,  Mtinch.  med.  Woch.,  1903. 

2  Milller,  Arch.  f.  Hyg.,  Ivi,  1906. 

3  Cap-ps  and  Miller,  Jour.  A.  M.  A.,  June,  1912,  p.  1848. 

^  Hamburger,  Bull,  of  the  Johns  Hopk.  Hosp.,  xxiv,  Jan.,  1913. 

5  Stokes  and  Weggefarth,  Med.  News,  91,  1897. 

^  Notter  and  Firth,  quoted  from  Harrington,  "Theory  and  Practice  of  Hygiene." 


BACTERIA  IN  MILK  707 

epidemic,  two  hundred  and  five  individuals  were  affected  with  vesic- 
lar  eruptions  of  the  throat,  with  tonsilHtis  and  swellings  of  the  cervical 
lymph  nodes.     Similar  cases  have  been  reported  by  Pott.^  > 

Although  anthrax  has  never  been  definitely  shown  to  have  been 
conveyed  by  milk,  Boschetti  ^  succeeded  in  isolating  hving  anthrax 
bacilli  from  a  sample  of  milk  two  weeks  after  its  withdrawal  from  the  cow. 

Milk  and  Tuberculosis. — The  question  of  the  conveyance  of  tuber- 
culosis by  means  of  milk  is  a  subject  which,  because  of  its  great  im- 
portance, has  been  extensively  investigated  by  bacteriologists.  A 
large  number  of  observers  have  succeeded  in  proving  the  presence  of 
tubercle  bacilli  in  the  milk  of  tuberculous  cows  by  intraperitoneal  in- 
oculation of  rabbits  and  guinea-pigs  with  samples  of  milk.  Such  posi- 
tive results  have  been  obtained  by  Bang,^  Hirschberger,"^  Ernst, ^  and 
many  others.  A  number  of  these  observers,  notably  Ernst,  have  shown 
that  tubercle  bacilli  may  be  present  in  the  milk  without  tuberculous  dis- 
ease of  the  udders.  In  an  examination  of  the  milk  supply  of  Washington, 
D.  C.,^  6.72  per  cent  of  the  samples  contained  tubercle  baciUi. 

The  path  of  entrance  of  the  bacilli  from  the  cow  into  the  milk  has 
long  been  a  subject  of  controversy.  That  the  bacilli  may  easily  enter 
the  milk,  when  tuberculous  disease  of  the  udder  is  present,  stands  to 
reason  and  is  universally  conceded.  It  is  now  believed  also,  on  the 
basis  of  much  experimentation,  that  in  systemically  infected  cows 
tubercle  bacilli  may  pass  through  the  mammary  glands  into  the  milk, 
without  evidence  of  local  disease  in  the  secreting  gland.  An  experi- 
ment performed  by  the  Royal  British  Tuberculosis  Commission  ^  illus- 
trates this  point.  A  cow,  injected  subcutaneously  with  tubercle  bacilli 
behind  the  shoulder,  began  to  discharge  tubercle  bacilli  in  the  milk 
within  seven  days  after  inoculation  and  continued  to  do  so  until  death 
from  generalized  tuberculosis. 

Milk  may  become  indirectly  contaminated,  furthermore,  with 
tubercle  bacilli  emanating  from  the  feces  of  cows.  It  has  been  shown 
that  tubercle  bacilli  are  present  in  the  feces  of  cattle  so  early  in  the 
disease  that  diagnosis  can  be  made  only  by  a  tubercuUn  test.^ 

Whether  or  not  contaminated  milk  is  common  as  an  etiological 

1  Pott,  Munch,  med.  Woch.,  1899.  ^  Boschetti,  Giorn.  med.  vet.,  1891. 

3  Bang,  Deut.  Zeit.  f .  Tierchem.,  xi,  1884. 

^  Hirschberger,  Deut.  Arch,  f .  klin.  Med.,  xliv,  1889. 

5  Ernst,  H.  C,  Amer.  Jour.  Med.  Sci.,  xcviii,  1890. 

6  Anderson,  BuU.  No.  41,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash.,  1908. 

7  Quoted  from  Mohler,  P.  H.,  and  Mar.  Hosp.  Serv.  Bull.  41,  1908. 
•  Schroeder  and  Cotton,  Bull.  Bureau  Animal  Industry,  Wash.,  1907. 


70^  BACTERIA  m  AIR,  SOIL,  WATER,  AND  MlLK 

factor  in  human  tuberculosis,  must  be  con^dered  at  present  as  an  ui*- 
settled  question.  Behring,  at  the  Congress  of  Veterinary  Medicine, 
at  Cassel,  in  1903,  advanced  the  view  that  pulmonary  tuberculosis  in 
adults  may  be  a  late  manifestation  of  a  milk  infection  contracted  dur- 
ing infancy.  He  stated  as  his  own  opinion,  moreover,  that  most  cases 
of  tuberculosis  in  man  are  traceable  to  this  origin.  The  problem  is  as 
difficult  of  solution  as  it  is  important.  In  bottle-fed  infants,  infection 
by  means  of  milk  unquestionably  occurs  with  considerable  frequency. 
Smith,^  Kossel,  Weber,  and  Huess,^  and  others,  have  isolated  tubercle 
bacilli  of  the  bovine  type  from  the  mesenteric  lymph  nodes  of  many 
infected  children.  Animal  experimentation  has,  furthermore,  revealed 
that  lesions  in  the  mesenteric  nodes,  as  well  as  later  in  the  bronchial 
lymph  nodes,  may  occur  as  a  consequence  of  feeding  tubercle  bacilli, 
without  any  demonstrable  lesions  in  the  intestinal  mucosa.  It  is  thus 
certain  that  infection  by  the  ingestion  of  tuberculous  milk  may  occur, 
especially  among  young  children  who,  as  is  well-known,  are  com- 
paratively susceptible  to  bacilli  of  the  bovine  type.  Whether  or  not  such 
infection  will  account  for  many  cases  of  tuberculosis  in  adults  is  a  ques- 
tion which,  for  final  solution,  will  require  much  more  investigation.  The 
sole  reliable  method  of  approaching  it  lies  in  determining  the  type, 
human  or  bovine,  of  the  bacilli  present  in  a  large  number  of  cases.  Ex- 
perience thus  far  seems  to  indicate  that  the  bovine  type  is  comparatively 
rare  in  the  pulmonary  disease  of  adults. 

The  value  of  the  tuberculin  reaction  for  diagnosis,  and  the  elimination 
of  all  cattle  showing  a  positive  reaction,  for  the  prevention  of  tubercu-  , 
losis,  can  not  be  overestimated.  The  failure  of  the  test  in  diseased 
animals  is  rare,  and  an  accurate  diagnosis  can  be  established  in  over 
90  per  cent  of  diseased  animals.^  The  assertion  that  the  cattle  are 
permanently  injured  by  tuberculin  injections  is  without  scientific  basis. 
If  this  test  were  conscientiously  carried  out,  and  infected  cattle  elim- 
inated, the  dangers  from  bovine  bacillus  infection  would  be  practically 
eliminated,  for  there  are  but  few  instances  in  which  science  has  been  able 
to  furnish  such  definite  information  for  absolute  protection.  It  is  need- 
less to  say,  however,  that  the  carrying  out  of  such  precautions  is 
subject  to  great  expense  and  great  difficulties  of  organization. 

Dairy  inspection  is  practiced  in  the  vicinity  of  many  of  our  larger 

1  Smith,  Trans.  Assn.  Amer.  Physic,  18,  1903. 

^Kossel,  Weber,  and  Huess,  Tuberkul.  Arb.    a.  d.  kais.  Gesundheitsamt,  1904, 
1905,  Hft.  1  and  3. 
» MoMer,  loc.  cit. 


BACTERIA  IN  MILK  709 

cities,  and  the  movement  is  daily  gaining  ground.  Until  fully  estab- 
lished, however,  upon  a  financial  basis  which  brings  the  best  products 
within  the  means  of  the  poorer  classes,  other  inexpensive  measures  to 
render  milk  safe  must  often  be  resorted  to. 

Sterilization  by  high  temperatures  is  objected  to  by  pediatricians 
because  of  the  physical  and  chemical  changes  produced  in  the  milk 
which  are  said  to  detract  from  its  nutritive  value. 

The  development  of  scurvy  and  rickets  in  infants  has  often  been 
attributed  to  the  use  of  such  milk.  These  objections,  however,  do  not 
apply  to  the  use  of  milk  which  has  been  subjected  to  the  process  of 
"pasteurization."  By  this  term  is  meant  the  heating  of  any  substance 
to  60°  C.  for  twenty  to  thirty  minutes.  The  process,  first  devised  by 
Pasteur  for  the  purpose  of  destroying  germs  in  wine  and  beer  in  which 
excessive  heating  was  supposed  to  injure  flavor,  brings  about  the  death 
of  all  microorganisms  which  do  not  form  spores — in  other  words,  of  all 
the  bacteria  likely  to  be  found  in  milk  which  can  give  rise  to  infection 
per  OS.  At  the  same  time  the  chemical  and  physical  constitution  of  the 
milk  is  not  appreciably  changed,  at  least  not  to  an  extent  which  renders 
it  less  valuable  as  a  food.  Statistics  by  Park  and  Holt  ^  have  shown 
strikingly  the  advantages  of  pasteurized  over  raw  milk  in  infant  feed- 
ing. Of  fifty-one  children  fed  with  raw  milk  during  the  summer  months, 
thirty-three  had  diarrhea,  two  died,  and  only  seventeen  remained 
entirely  well.  Of  forty-one  receiving  pasteurized  milk,  but  ten  had 
diarrhea,  one  died,  and  thirty-one  remained  entirely  well  throughout 
the  summer.  The  actual  diminution  of  the  living  bacterial  contents 
of  milk  by  pasteurization  is  enormous,  the  milk  so  treated  often  con- 
taining not  more  than  one  thousand,  usually  less  than  fifteen  thou- 
sand, living  bacteria  to  each  cubic  centimeter. 

Methods  of  Estimating  the  Number  of  Bacteria  in  Milk. — In  estimating 
the  number  of  bacteria  in  milk,  colony  counting  in  agar  or  gelatin  plates 
is  resorted  to.  Great  care  must  be  exercised  in  obtaining  the  specimens. 
If  taken  from  a  can,  the  contents  of  the  can  should  be  thoroughly  mixed, 
since  the  cream  usually  contains  many  more  bacteria  than  the  rest  of 
the  milk.  The  specimen  io  then  taken  into  a  sterile  test  tube  or  flask. 
If  the  milk  is  supplied  in  an  ordinary  milk  bottle,  this  should  be 
thoroughly  shaken  before  being  opened,  and  the  specimen  for  exam- 
ination taken  out  with  a  sterile  pipette.  Dilutions  of  the  specimen  can 
then  be  made  in  sterile  broth  or  salt  solution.     If  an  initial  dilution 

» Park  and  Holt,  loc.  cit. 


710  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

of  1  :  100  is  made,  quantities  ranging  from  1  c.c.  to  0.1  c.c.  of  this  will 
furnish  0.01  c.c.  to  0.001  c.c.  of  the  milk,  respectively.  Inoculation  of 
properly  cooled  tubes  of  melted  neutral  agar  and  gelatin,  with  varying 
quantities  of  these  dilutions,  are  then  made  and  plates  poured.  After 
twenty-four  to  forty-eight  hours  at  room  temperature  or  in  the  in- 
cubator, colony  counting  is  done  as  described  upon  page  161,  and  the 
proper  calculation  is  made.  In  samples  in  which  few  bacteria  are  ex- 
pected, direct  transference  of  1/20  or  1/40  of  a  c.c.  of  the  whole  milk 
into  the  agar  may  be  made.  This  method  saves  time  but  is  less  accurate. 

Direct  Methods  of  Counting  Bacteria. — Direct  methods  of  counting 
bacteria  in  milk  have  recently  been  advised,  the  one  most  extensively 
tried  being  that  of  Prescott  and  Breed.  By  this  method  a  capillary 
tube  is  marked  to  measure  accurately  0.01  c.c.  This  amount  of  the 
milk  is  spread  over  a  square  cm.  on  a  microscope  slide.  It  is  dried  in 
the  air  and  fixed  with  methyl  alcohol,  after  which  the  fatty  constituents 
can  be  dissolved  with  xylol.  It  can  then  be  stained  lightly  with  the  Jen- 
ner  stain.  The  bacteria  are  counted  under  an  oil  immersion  lens,  the 
tube  length  and  magnification  being  so  arranged  that  the  microscopic 
field  covers  1/50  sq.  mm.  A  standardized  eyepiece  micrometer  may  be 
used.  The  average  number  of  bacteria  found  in  such  fields  may  be 
multiplied  by  5,000  to  give  the  number  of  bacteria  contained  in  0.01 
c.c.  of  milk.  This  method  has  not  yet  displaced  the  one  of  plating 
and  does  not  promise  to  do  so  for  some  time. 

For  the  isolation  of  special  pathogenic  bacteria  from  milk,  no  rules 
can  be  laid  down,  since,  in  every  case,  the  method  adapted  to  the. par- 
ticular organism  sought  for  must  be  chosen. 

Tubercle  bacilli  can  be  isolated  from  milk  with  success  only  by 
guinea-pig  injection.  The  milk  is  centrifugalized  and  5  c.c.  of  the 
sediment,  together  with  some  of  the  cream  that  has  risen  to  the  top, 
is  intraperitoneally  or  subcutaneously  injected. 

The  control  of  milk  in  the  market  depends  upon  careful  legulations, 
which  must  include  care  of  cattle,  dairy  inspection  and  bacteriological 
control  of  the  delivered  milk.  This  is  a  subject  which  is  too  extensive 
to  touch  upon  in  a  book  of  this  kind.  However,  a  general  idea  of  the 
methods  employed  may  be  obtained  by  studying  the  accompanying 
table,  which  is  taken  from  the  New  York  City  Department  of  Health 
Regulation  for  the  Sale  of  Milk  and  Cream. 

Bacteria  and  Butter. — Butter  is  made  from  cream  separated  from 
milk  either  by  standing  or  by  centrifugalization.  After  this,  the  cream 
is  agitated  by  churning,  which  brings  the  small  fat-globules  into  mutual 


BACTERIA  IN  MILK  711 

contact,  allows  them  to  adhere  to  each  other  and  form  clumps  of  butter. 
It  has  been  a  matter  of  common  experience,  however,  that  unless  the 
cream  is  allowed  to  ** ripen"  for  a  considerable  period  before  churning, 
the  resulting  butter  lacks  the  particular  quality  of  flavor  which  gives  it 
its  market  value.  The  interval  of  ripening,  at  first  a  necessity  upon 
small  farms  where  cream  must  be  collected  and  allowed  to  accumulate, 
has  now  been  recognized  as  an  essential  for  the  production  of  the  best 
grades  of  butter,  and  it  has  been  shown  that  the  changes  taking  place 
in  the  cream  during  this  period  are  referable  to  the  action  of  bacteria. 
Cream,  which  before  the  ripening  process  contains  but  50,000  bacteria 
to  each  cubic  centimeter,  at  the  end  of  a  period  of  "ripening"  will  often 
contain  many  millions  of  microorganisms.  At  the  same  time,  the  cream 
becomes  thick  and  often  sour. 

The  species  of  bacteria  which  take  part  in  this  process  and  which, 
therefore,  must  determine  to  a  large  extent  the  quality  of  the  end  prod- 
uct, are  various  and,  as  yet,  incompletely  known.  Usually  some  variety 
of  lactic-acid  bacilli  is  present  and  these,  as  in  milk,  outgrow  other  species 
and,  according  to  Conn,^  are  probably  essential  for  "ripening." 

It  would  be  of  great  practical  value,  therefore,  if  definite  pure  cul- 
tures of  the  bacteria  which  favor  the  production  of  agreeable  flavors 
could  be  distributed  among  dairies.  In  Denmark  this  has  been  attempt- 
ed by  first  pasteurizing  the  cream  and  then  adding  a  culture  of  bacteria 
isolated  from  "favorable"  cream.  These  cultures,  delivered  to  the 
dairyman,  are  planted  in  sterilized  milk,  in  order  to  increase  their  quan- 
tity, and  this  culture  is  then  poured  into  the  pasteurized  cream.  In 
most  cases,  these  so-called  "starters"  are  not  pure  cultures,  but  mix- 
tures of  three  or  more  species  derived  from  the  original  cream. 

Adverse  accidents  in  the  course  of  butter-making,  such  as  "souring" 
or  "bittering"  of  butter,  -are  due  to  the  presence  of  contaminating, 
probably  proteolytic,  microorganisms  in  the  cream  during  the  process  of 
"ripening." 

As  a  means  of  transmitting  infectious  disease,  butter  is  of  importance 
only  in  relation  to  tuberculosis.  Obermuller,^  Rabinowitch,^  Boyce,'^ 
and  others,  have  repeatedly  found  tubercle  bacilli  in  market  butter,  and 
Mohler,^  Washburn,  and  Rogers  have  recently  shown  that  these  bacilli 

1  Crnin,  "Agricultural  Bacteriology,"  Phila.,  1901. 

2  Obermiiller,  Hyg.  Rundschau,  14,  1897. 
'  Rabinotoitch,  Zeit.  f.  Hyg.,  xxvi,  1897. 

*  Boyce  and  Woodhead,  Brit.  Med.  Jour.,  2,  1897. 

»  Mohler,  U.  S.  P.  H.  and  Mar.  Hosp.  Serv.  Bull.  41,  1908. 


712  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

REGULATIONS  GOVERNING  THE  GRADES  AND  DESIGNATION  OF  MILK 
The  following  classifications  apply  to  milk  and  cream.    The  regulations  regarding 


m 


GRADES  OF 
MILK  OR 
CREAM 
WHICH  MAY 
BE  SOLD  IN 
THE  CITY  OF 
NEW.YORK 

DEFINITION 

TUBERCULIN 
TEST  AND 
PHYSICAL 
CONDITION 

BACTERIAL  CONTENTS 

GRADE  A 

Milk  or  Cream 
(Raw) 

Grade  A  milk  or  cream  (raw)  is  milk  or 
cream  produced  and  handled  in  accord- 
ance with  the  minimum  requirements, 
rules  and  regulations  as  herein  set  forth. 

1.  Only  such 
cows  shall  be  ad- 
mitted to  the 
herd  as  have  not 
reacted  to  a 
diagnostic  injec- 
tion of  tubercu- 
lin and  are  in 
good  physical 
condition. 

2.  All    cows 
shall  be  tested 
annually     with 
tuberculin    and 
all  reacting  ani- 
mals shall  be  ex- 
cluded from  the 
herd. 

Grade  A  milk  (Raw)  shall  not  con- 
tain more  than  60,000  bacteria  per 
c.c.  and  cream  more  than  300,000 
bacteria  per  c.c.  when  delivered  to 
the  consumer  or  at  any  time  prior  to 
such  delivery. 

Milk  or  Cream 
(Pasteurized) 

Grade  A  milk  or  cream  (pasteurized)  is 
milk  or  cream  handled  and  sold  by  dealers 
holding  permits  therefor  frpm  the  Board 
of  Health,  and  produced  and  handled  in 
accordance  with  the  requirements,  rules 
and  regulations  as  herein  set  forth. 

No  tuberculin 
test  required  but 
cows    must    be 
healthy  as  dis- 
closed by  physi- 
cal examination 
made  annually. 

Grade  A  milk  (pasteurized)  shall 
not  contain  more  than  30,000  bac- 
teria per  c.c.  and  Cream  (pasteurized) 
more  than  150,000  bacteria  per  c.c. 
when  delivered  to  the  consumer  or  at 
any  time  after  pasteurization  and 
prior  to  such  delivery. 

No  milk  supply  averaging  more 
than  200,000  bacteria  per  c.c.  shall 
be  pasteurized  for  sale  under  this 
designation. 

GRADE  B 

Milk  or  Cream 
(Pasteurized) 

Grade  B  milk  or  cream  (pasteurized)  is 
milk  or  cream  produced  and  handled  in 
accordance  with  the  minimum  require- 
ments, rules  and  regulations  herein  set 
forth  and  which  has  been  pasteurized  in 
accordance  with  the  requirements  and 
rules  and  regulations  of  the  Department 
of  Health  for  pasteurization. 

No  tuberculin 
test  required  but 
cows    must    be 
healthy  as  dis- 
closed by  physi- 
cal examination 
made  annually. 

No  milk  under  this  grade  shall 
contain  more  than  100,000  bacteria 
per  c.c.  and  no  cream  shall  contain 
more  than  500,000  bacteria  per  c.c. 
whep  delivered  to  the  consumer  or  at 
any  time  after  pasteurization  and 
prior  to  such  delivery.                      * 

No  milk  supply  averaging  more 
than  1,500,000  bacteria  per  c.c.  shall 
be  pasteurized  in  this  city  for  sale 
under  this  designation. 

No  milk  supply  averaging  more 
than  300,000  bacteria  per  c.c.  shall  be 
pasteiu-ized  outside  of  this  city  for 
sale  under  this  designation. 

GRADE  C 
Milk  or  Cream 

(Pasteurized) 
(For  cooking 
and     manu- 
facturing    pur- 
poses only). 

Grade  C  milk  or  cream  is  milk  or  cream 
not  conforming  to  the  requirements  of 
any  of  the  subdivisions  of  Grade  A  or 
Grade  B  and  which  has  been  pasteiu-ized 
according  to  the  requirements  and  rules 
and  regulations  of  the  Board  of  Health  or 
boiled  for  at  least  two  (2)  minutes. 

No  tuberculin 
test  required  but 
cows    must    be 
healthy  as  dis- 
closed by  physi- 
cal examination 
made  annually. 

No  milk  of  this  grade  shall  contain 
more  than  300.000  bacteria  per  c.c. 
and  no  cream  of  this  grade  shall  con- 
tain more  than  1,500,000  bacteria 
per  c.c.  after  pasteurization. 

NOTE — Sour  milk,  buttermilk,  sour  cream,  kumyss,  matzoon,  zoolac  and  similar  products  shall  not  be  made 
the  process  of  souring.    Sour  cream  shall  not  contain  a  less  percentage  of  fats  than  that  designated  for  cream. 

No  other  words  than  those  designated  herein  shall  appear  on  the  label  of  any  container  containing  milk  or  cream 

The  term  "  certified  "  milk  is  usually  defined  for  each  region  by  a  special  commission  of  the  County 
Milk  Commission  of  Kings  County,  N.  Y.: 

Certified  Milk  must  have  every  characteristic  of  pure,  clean,  fresh,  wholesome  cow's  milk.  The 
or  preservatives.     Nothing  must  be  added  to  the  milk  and  nothing  taken  away. 

Certified  Milk  shall  not  contain  less  than  4  per  cent  of  butter  fat. 

» Table  taken  from  Rules  and  Regulations  of  N.  Y.  City  Department  of  Health,  1914,  applying  to 


BACTERIA  IN  MILK 

AND  CREAM  WHICH  MAY  BE  SOLD  IN  THE  CITY  OF  NEW  YORRi 
bacterial  content  and  time  of  delivery  shall  not  apply  to  sour  cream 


713 


NECES- 
SARY 
SCORES 

FOR 
DAIRIES 
PRODUC- 
ING 

TIME  OF 
DELIVERY 

BOTTLING 

LABELING 

PASTEUR. 
IZATION 

Equip.  25 
Meth.  50 
Total  75 

Shall   be   de- 
livered    within 
36   hours   after 
production. 

Unless     other- 
wise   specified    in 
the    permit    this 
milk      or     cream 
shall  be  delivered 
to    the  consumer 
only  in  bottles. 

Outer  caps  of  bottles  shall  be  white  and 
shall  contain  the  words  Grade  A,  Raw, 
in  black  letters  in  large  type,  and  shall 
state  the  name  and  address  of  the  dealer. 

Equip.  25 
Meth.  43 
Total  68 

Shall   be   de- 
livered    within 
36   hours   after 
pasteurization. 

Unless      other- 
wise   specified   in 
the     permit    this 
milk     or     cream 
shall  be  delivered 
to   the   consumer 
only  in  bottles. 

Outer  caps  of  bottles  shall  be  white  and 
shall  contain  the  words  Grade  A  in  black 
letters  in  large  type,  date  and  hours  be- 
tween which  pasteurization   was  com- 
pleted; place  where  pasteurization  was 
performed;  name  of  the  person,  firm  or 
corporation  offering  for  sale,  selling  or 
delivering  same. 

Only  such 
milk  or  cream 
shall  be  re- 
garded as  pas- 
teurized as  has 
been  subjected 
to  a  tempera- 
ture averaging 
145°  Fahr.  for 
not  less  than  30 
minutes. 

Equip.  20 
Meth.  35 
Total  55 

Milk  shall  be 
delivered  within 
36    hours    and 
cream  within  48 
hours  after  pas- 
teurization. 

May   be   deliv- 
ered   in    cans    or 
bottles. 

Outer  caps  of  bottles  containing  milk 
and  tags  affixed  to  cans  containing  milk 
or   cream    shall  be  white  and  marked 
"Grade  B"  in  bright  green  letters  in 
large  type,  date  pasteurization  was  com- 
pleted, place  where  pasteurization  was 
performed,  name  of  the  person,  firm  or 
corporation  offering  for  sale,  selling  or  de- 
livering same.    Bottles  containing  creams 
shall  be  labeled  with  caps  marked  "Grade 
B"  in  bright  green  letters,  in  large  type 
and  shall  give  the  place  and  dat«  of  bot- 
tling and  shall  give  the  name  of  person, 
firm  or  corporation  offering  for  sale,  sell- 
ing or  delivering  same. 

Only  such 
milk  or  cream 
shall  be  re- 
garded as  pas- 
teurized as  has 
been  subjected 
to  a  tempera- 
ture averaging 
145°  Fahr.  for 
not  less  than  30 
minutes. 

Score  40 

• 

Shall  be  de- 
livered    within 
48   hours   after 
pasteurization. 

May  be   deliv- 
ered in  cans  only. 

Tags  affixed  to  cans  shall  be  white  and 
shall  be  marked  in  red  with  the  words 
"Grade  C"  in  large  type  and  "for  cook- 
ing" in  plainly  visible  type,  and  cans  shall 
have  properly  sealed  metal  collars,  paint- 
ed red  on  necks. 

Only  such 
milk  or  cream 
shall  be  re- 
garded as  pas- 
teurized as  has 
been  subjected 
to  a  tempera- 
ture averaging 
145°  Fahr.  for 
not  less  than  30 
minutes. 

from  any  milk  of  a  less  grade  than  that  designated  for  "  Grade  B  "  and  shall  be  pasteurized  before  being  put  through 

or  milk  or  cream  products  except  the  word  "certified"  when  authorized  under  the  State  laws. 

Med.  Soc,  sanctioned  by  State  Law.    The  following  is  the  definition  of  certified  milk  given  by  the 

milk  must  be  in  its  natural  state,  not  having  been  heated  and  without  the  addition  of  coloring  matter 
sale  of  milk  and  cream. 


714  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

could  remain  alive  and  virulent  for  as  long  as  five  months  in  butter  kept 
at  refrigerator  temperature.  The  acid-fast  butter  bacillus,  described  by 
Rabinowitch  as  similar  to  the  true  Bacillus  tuberculosis,  shows  decided 
cultural  and  morphological  differences  from  the  latter. 

Bacteria  and  Cheese. — The  conversion  of  milk  products  into  cheese 
consists  in  a  process  of  proteid  decomposition  which,  by  its  end  products, 
leucin,  tyrosin,  and  ammonia  compounds,  largely  determines  the  cheese- 
flavors.  The  production  of  cheese,  therefore,  is  due  to  the  action  of 
proteolytic  bacterial  enzymes  ^  and  the  variety  of  a  cheese  is  largely 
determined  by  the  microorganisms  which  are  present  and  by  the  cul- 
tural conditions  prevailing.  The  sterilization  of  cream,  or  the  addition 
of  antiseptics,  absolutely  prevents  cheese  production. 

The  organisms  which  are  concerned  in  such  processes  have  been  ex- 
tensively studied  and  attempts  have  been  made,  with  moderate  success, 
to  produce  a  definite  flavor  with  pure  cultures. 

In  the  production  of  cheese  the  two  varieties,  hard  and  soft  cheeses, 
depend  not  so  much  upon  the  bacterial  varieties  as  upon  the  differences 
in  the  treatment  of  the  curds  before  bacterial  action  has  begun.  In  the 
former  case,  a  complete  freeing  of  the  curds  from  the  whey  furnishes  a 
culture  medium  which  is  comparatively  dry  and  of  almost  exclusively 
proteid  composition;  in  the  latter,  retention  of  whey  gives  rise  to  cul- 
tural conditions  in  which  more  rapid  and  complete  bacterial  action  may 
take  place.  The  holes,  which  are  so  often  observed  in  some  of  the  hard 
cheeses,  are  due  to  gas  production  during  the  process  of  ''ripening." 

As  to  the  varieties  of  microorganisms  present  in  various  cheeses,  much 
careful  work  has  been  done.  Duclaux  ^  attributed  the  ''ripening"  of 
some  of  the  soft  cheeses  to  a  microorganism  closely  related  to  Bacillus 
subtilis.  V.  Freudenreich  ^  in  part  substantiated  this,  but  laid  particular 
stress  upon  the  action  of  Oi'dium  lactis,  a  mold,  and  upon  several  vari- 
eties of  yeast.  Conn,^  more  recently,  in  a  bacteriological  study  of  Cam- 
embert  cheese,  has  demonstrated  that  the  production  of  this  cheese 
depends  upon  the  united  action  of  two  microorganisms,  one  an  oidium, 
like  the  Oidium  lactis  of  Freudenreich,  which  is  found  chiefly  in  the 
interior  softened  areas,  the  other  a  mold  belonging  to  the  penicillium 
variety,  found  in  a  matted  felt-work  over  the  surface  and  penetrating 
but  a  short  distance.     In  spite  of  the  scientific  basis  upon  which  the 

1  Freudenreich,  Koch's  Jahresbericht,  etc.,  135,  1891. 

2  Duclaux,  "Le  Lait,"  Paris,  1887. 

3  V.  Freudenreich,  Cent.  f.  Bakt.,  II,  i,  1895. 

*  Conn,  BuU.  Statis.  Agri.  Exp.  Stat.  35,  1905. 


LACTIC-ACID  BACILLI  7lS 

work  of  these  men  and  of  others  has  seemed  to  place  cheese  production, 
attempts  at  uniformity  in  cheese  production  have  met  with  almost  in- 
superable obstacles  because  of  the  presence  of  a  variety  of  adventitious 
microorganisms  which,  depending  in  species  and  proportion  upon  the 
local  conditions  under  which  the  various  cheeses  have  been  produced, 
have  added  minor  characteristics  of  flavor  which  have  determined  mar- 
ket value.  Occasional  failure  of  good  results  in  cheese  production  ^  is 
due  to  contamination  with  other  chromogenic  or  putrefactive  bacteria. 
In  its  relationship  to  the  spread  of  infectious  disease,  cheese  is  rela- 
tively unimportant  except  in  regard  to  tuberculosis.  Typhoid  and 
other  non-spore  forming  pathogenic  germs  can  not  survive  the  condi- 
tions existing  during  cheese-ripening  for  any  length  of  time.  Tubercle 
bacilli,  both  of  the  human  and  bovine  types,  have  been  found  in  cheese 
by  Harrison  ^  and  others,  and  Galtier  has  shown  experimentally  that 
tubercle  bacilli  may  remain  alive  and  virulent  in  both  salted  and  un- 
salted  cheese  for  as  long  as  ten  days. 

THE   LACTIC-ACID   BACILLI  AND   METCHNIKOPP'S   BACTERIO- 

THERAPY 

A  problem  which  has  occupied  clinical  investigation  for  many  years 
is  that  of  gastrointestinal  autointoxication.  There  are  a  number  of 
conditions  occurring  in  man,  in  which  symptoms  profoundly  affecting 
the  nervous  system,  the  circulation,  and,  in  a  variety  of  ways,  the  entire 
body,  can  be  clinically  traced  to  the  intestines,  and  can,  in  many  cases, 
be  relieved  by  thorough  purgation  and  careful  diet.  In  some  of  these 
conditions,  specific  microorganisms  can  be  held  accountable  for  the 
diseases  (B.  enteritidis,  B.  botulinus,  etc.).  In  other  cases,  however, 
etiological  investigations  have  met  with  but  partial  success  because  of 
the  large  variety  of  microorganisms  present  in  the  intestinal  tract  and 
because  of  the  complicated  symbiotic  conditions  thereby  produced. 
Intestinal  putrefaction,  recognized  as  the  cardinal  feature  of  such 
maladies,  has  been  attributed  to  Bacillus  proteus  vulgaris,^  to  Bacillus 
aerogenes  capsulatus,  to  Bacillus  putrificus,^  and  to  a  number  of  other 
bacteria,  but  definite  and  satisfactory  proof  as  to  the  etiological  im- 
portance of  any  of  these  germs  has  not  yet  been  advanced.    The  fact 

1  Beijerinck,  Koch's  Jahresber,  etc.,  82,  189, 

2  Harrison  and  Galtier,  quoted  from  Mohler,  U.  S.  Pub.  H.  and  Mar.  Hosp.  Serv., 
Hygiene  Lab.  Bull.  41,  1908. 

'  Lesage,  Rev.  de  m^d.,  1887. 

*  Tissier.  Ann.  de  Tinst.  Pasteur,  1905. 


716  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

remains,  however,  that,  whatever  may  be  the  specific  cause,  the  disease 
itself,  a  grave  and  often  fatal  affliction,  may  be  clinically  traced,  in  a 
number  of  cases,  to  the  absorption  of  poisons  from  the  intestinal  canal, 
and  it  is  more  than  likely  that  these  poisons  are  the  products  of  bacterial 
activity.  Reason  dictates,  furthermore,  that  the  bacteria  primarily 
responsible  for  the  production  of  these  toxic  substances  do  not  belong  to 
the  varieties  which  attack  carbohydrates  only,  but  must  belong  to  that 
class  of  aerobic  and  anaerobic  germs  which  possess  the  power  of  breaking 
up  proteids — in  other  words,  the  bacteria  of  putrefaction. 

On  the  basis  of  the  mutual  antagonism  existing  in  culture  between 
many  acid-producing  bacteria  and  those  of  putrefaction — a  phenomenon 
recognized  by  some  of  the  earliest  workers  in  this  field,  many  investiga- 
tors have  suggested  the  possibility  of  combating  intestinal  putrefaction 
by  adding  acid-forming  bacteria  together  with  carbohydrates  to  the  diet 
of  patients  suffering  from  this  condition.  The  first  to  suggest  this 
therapy  was  Escherich  ^  who  proposed  the  use,  in  this  way,  of  Bacillus 
lactos  aerogenes;  with  the  same  end  in  view,  Quincke,^  a  little  later, 
suggested  the  use  of  yeasts — Oidiiun  lactis.  The  reasoning  underlying 
these  attempts  was  meanwhile  upheld  by  experiments  carried  out  both 
in  vitro  and  upon  the  living  patient.  Thus  Brudzinski  ^  was  able  to 
demonstrate  that  Bacillus  lactis  aerogenes,  in  culture,  inhibited  the 
development  of  certain  races  of  the  proteus  species  and  succeeded  in 
obtaining  markedly  favorable  results  by  feeding  pure  cultures  of  Bacillus 
lactis  aerogenes  to  infants  suffering  from  fetid  diarrhea.  Similar  ex- 
periments ^  carried  out  with  the  Welch  bacillus  (aerogenes  capsulatus) 
and  Bacillus  coli,  however,  had  no  such  corroboratory  results,  since  this 
anaerobe  possesses  a  considerable  resistance  against  an  acid  reaction. 
In  considering  the  difficulties  of  the  problems  involved  in  this  question, 
it  occurred  to  Metchnikoff  ^  that  much  of  the  practical  failure  of  therapy, 
based  upon  the  principles  stated  above,  might  be  referred  to  insufficient 
powers  of  acid  production  on  the  part  of  Bacillus  coli.  Bacillus  lactis 
aerogenes,  and  other  germs  previously  used.  In  searching  for  more  pow- 
erful acid  producers,  his  attention  was  attracted  to  Bacillus  bulgaricus, 

^  Escherich,  Therapeut.  Monatshefte.,  Oct.,  1887. 

2  Quincke,  Verhandl.  des  Congress  f .  Inn.  Med.,  Wiesbaden,  1898. 
•  ^  BrudJinski,  Jahrbuch  f.  Kinderheilkunde,  52,  1900  (Erganzungshef t) . 

^  Tissier  and  Martelly,  Ann.  de  I'inst.  Pasteur,  1906. 

^  Metchnikoff,  "Prolongation  of  Life,"  G.  P.  Putnam's  Sons,  N.  Y.;  also  in  "Bac- 
t^riotherapie,"  etc.  "  Bibliotheque  de  th^rapeutique,"  Gilbert  and  Carnot,  Paris, 
1909. 


BACTERIA  IN  THE  INDUSTRIES 


717 


isolated  from  milk  by  Massol  ^  and  Cohendy  ^  in  1905.  This  bacillus, 
according  to  the  researches  of  Bertrand  and  Weisweiller,^  produces  as 
much  as  25  grams  of  lactic  acid  per  liter  of  milk.  In  addition  to  this, 
it  manufactures,  from  the  same  quantity  of  milk,  about  50  centigrams 
of  acetic  and  succinic  acids  and  exerts  no  putrefactive  action  upon  pro- 


FiG.  155. — Bacillus  bulgarictjs. 

teids.    Added  to  these  characters,  it  is  especially  adapted  to  therapeutic 
appUcation  by  its  complete  lack  of  pathogenicity. 

The  administration  of  the  bacillus  to  patients  suffering  from  intestinal 
putrefaction,  first  suggested  by  Metchnikoff  in  1906,  has,  since  that  time, 
been  extensively  practiced  and  often  with  remarkable  success.  In  spite 
of  sharp  criticism,  especially  by  Luersen  and  Kiihn,^  who  deny  much  of 


1  Massol,  Revue  medicale  de  la  Suisse  romande,  1905. 

2  Cohendy,  Comptes  rend,  de  la  soc.  de  biol.,  60,  1906. 

'  Bertrand  and  Weisweiller,  Ann.  de  I'inst.  Pasteur,  1906. 
*  Luersen  and  Kilhn,  Cent,  f .  Bakt.,  II,  xx,  1908. 


718  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

the  antiputrefactive  activity  of  the  bacillus,  the  treatment  of  Metch- 
nikoff  has  found  many  adherents,  upon  the  basis  of  purely  clinical  ex- 
periment. It  is  not  possible  to  review  completely  the  already  extensive 
literature.  Among  the  more  valuable  contributions  may  be  mentioned 
the  articles  by  Grekoff,^  by  Wegele,^  and  by  Klotz.^  In  Metchnikoff's 
experinients  and  in  the  work  of  his  immediate  successors,  the  bacillus 
was  used  either  in  milk  culture  or  in  broth  in  which  it  was  induced  to 
grow  in  symbiosis  with  other  microorganisms. 

Recently,  North  ^  has  suggested'^^he  use  of  Bacillus  bulgaricus  in 
parts  of  the  body  other  than  the  digestive  tract.  His  work  was  made 
feasible  by  the  discovery  that  the  bacillus  could  be  cultivated  in  dex- 
trose-pepton  broth  to  which  calcium  carbonate  has  been  added,  after 
the  manner  recommended  by  Hiss.  With  such  cultures,  applied  in  the 
form  of  a  spray,  inflammations  of  the  ear,  nose,  throat,  genitourinary 
tract,  etc.,  have  been  treated,  many  of  them  with  success. 

BACTERIOLOGICAL  EXAMINATION  OF  OYSTERS 

On  accoimt  of  the  danger  of  the  transmission  of  typhoid  fever  by. 
oysters  which  have  been  bred  or  stored  in  contaminated  water,  stand- 
ard methods  ^  have  been  devised  for  the  estimation  of  the  bacterial  con- 
tent of  oysters.  These  are  similar  in  principle  and  method  to  those 
used  for  the  examination  of  water,  and  a  most  important  index  of 
sewage  contamination  and  consequent  danger  of  typhoid  infection  is 
*the  number  of  colon  bacilli  present  in  the  shell  fish.  The  shell  liquor 
is  used  for  examination,  and  in  examining  oysters  in  the  shell  the  fol- 
lowing procedure  is  followed:  Five  oysters  having  deep  bowls  and 
closed  shells  are  selected.  Lips  of  the  shell  are  sterilized  in  the  flame 
or  by  burning  with  alcohol.  The  liquor  is  obtained  by  opening  the 
shell  with  a  sterilized  knife,  or  better,  by  drilling  a  hole  through  the 
flame  surface  with  a  sterilized  gimlet.  For  determining  the  total  num- 
ber of  bacteria  the  shell  liquor  is  withdrawn  with  a  sterilized  pipette, 
diluted  with  1  per  cent  salt  solution,  and  placed  in  agar,  as  described 
on  page  693.  More  important,  however,  is  the  presumptive  colony 
test,  which  is  carried  out  by  inoculating  three  lactose  bile  tubes  with 

^Grekoff,  "Observations  cliniques  sur  I'effet  du  lact.  agri.,"  etc.,  St.  Petersburg, 
1507. 

^  Wegele,  Deut.  med.  Woch.,  xxxiv,  1908. 
'  Klotz,  Zentralbl.  f.  innere  Med.,  1908. 
*  North,  Med.  Record,  March,  1909. 
»  Amer.  Jour.  Pub.  Health,  1913,  ii,  34. 


1 


EXAMINATION  FOR  BACTERIA  IN  OYSTERS  719 

1.0  c.c,  0.1  c.c,  and  0.01  c.c,  respectively,  from  each  of  the  five  oysters. 
The  tubes  are  incubated  for  three  days,  and  the  development  of  over 
10  per  cent  of  gas  in  the  closed  arm  is  considered  a  positive  reaction. 
The  score  is  recorded  as  the  approximate  number  of  colon  bacilli  con- 
tained in  the  5.55  c.c.  of  shell  liquor  from  the  five  oysters,  and  is  esti- 
mated in  the  following  way :  A  positive  reaction  in  a  tube  inoculated 
in  1  c.c.  is  recorded  as  1.0,  a  positive  reaction  in  0.1  c.c,  is  10,  and 
in  0.01  is  recorded  as  100.  The  sum  of  these  figures  is  the  score  for 
the  batch  of  oysters  from  which  the  five  have  been  taken.  In  examin- 
ing shucked  oysters  a  well-mixed  sample  of  oysters  and  the  surround- 
ing fluid  are  put  in  a  sterilized  vessel  and  lactose  bile  tubes  inoculated 
in  triplicate  with  1.0  c.c,  0.1  c.c,  0.01  c.c,  0.001  c.c  of  the  liquor. 
No  definite  standard  score  has  been  adopted,  but  the  United  States 
Pure  Food  Board  ^  has  condemned  unshucked  stock  having  a  score  of 
32  or  higher. 

BACTERIA   IN   THE   INDUSTRIES 

Bacteria  and  Tobacco. — In  the  manufacture  of  tobacco,  the  har- 
vested leaves  are  first  dried  and  then  heaped  up  in  large  masses,  in 
which  the  tobacco  undergoes  fermentation.  During  this  fermentation, 
which  goes  on  at  temperatures  varying  from  50°  C.  to  60°  C,  carbohy- 
drates are  split  up  and  much  nicotin  is  destroyed.^  The  end  products 
consist  largely  of  CO2  and  various  organic  acids,  butyric,  formic, 
succinic,  etc  During  the  fermentation,  bacteria  of  many  varieties  are 
found  in  the  heaps  of  tobacco  leaves  and  many  attempts  have  been 
made  to  determine  flavors  artificially  by  inoculating  tobacco  leaves  of 
a  poorer  quality  with  cultures  obtained  from  the  finer  Havana  grades. 
Suchsland  ^  and  others,  who  have  attempted  this,  claim  to  have  ob- 
tained marked  improvements  in  domestic  products  by  this  method. 
The  bacteria  found  in  tobacco  fermentation  belong  to  many  varieties. 
Some  of  these  are  closely  related  to  the  proteus  and  subtilis  groups. 
Others  are  distinctly  thermophilic,  an  attribute  required  by  the  high 
temperatures  attained  in  the  fermenting  tobacco  leaves.  It  is  probable 
that  the  tobacco  flavors  cannot  be  regulated  by  bacteriological  methods 
alone,  since  it  has  been  shown  by  Loew  *  that  an  important  factor  in  the 

1  Gorham,  U.  S.  Pure  Food,  Amer.  Jour.  Pub.  Health,  1913,  ii,  32. 

^  Behrens,  quoted  from  Flugge,  "Die  Mikroorganismen,"  Bd.  1,  Leipzig,  1896, 

'  Suchsland,  Ber.  der  Deut.  botan.  Ges.,  ix. 

<  Loew,  Rep.  U.  S.  Dep.  Agriculture,  59,  1899, 


720  BACTERIA  IN  AIR,  SOIL,  WATER,  AND  MILK 

tobacco  fermentation  is  contributed  by  the  leaf-enzymes,  which,  of 
course,  depend  intimately  upon  soil  and  climatic  conditions. 

Opium  Productions. — In  the  preparation  of  opium  f oi*  smoking  pur- 
poses, the  raw  product  is  subjected  to  a  prolonged  period  of  fermenta- 
tion by  which  the  carbohydrates  in  the  material  are  destroyed.  Accord- 
ing to  various  observers,  the  process  is  carried  out  in  most  cases  by  a 
species  of  aspergillus. 

Indigo  Production. — Indigo,  which  is  obtained  from  the  plants 
''Isatis  tinctoria*'  and  ''Indigofer  tinctoria,"  is  not  present,  as  such, 
in  the  plants.  In  some  of  these  it  is  found  in  the  form  of  indican,  in 
others,  as  indoxyl.  It  has  been  shown  by  Alvarez  that  the  oxidation  of 
indican  and  indoxyl  into  indigo-blue  is  carried  out  largely  by  bacterial 
oxydases.  Sterilized  indigo  plants  do  not  produce  the  blue  color.  Alva- 
rez ^  has  isolated  a  bacillus  closely  related  to  the  Bacillus  mucosus 
capsulatus  group,  to  the  action  of  which  he  attributes  this  oxidation. 

Bacteria  in  the  Tanning  of  Hides. — Raw  animal  hides  are  subject 
to  decomposition  until  treated  by  a  process  known  as  tanning.  This 
consists  first  in  the  depilation  of  the  dried  and  salted  skins,  either  by 
partial  putrefaction  in  an  atmosphere  saturated  with  water  vapor  or 
by  chemical  treatment  with  solutions  of  milk  of  lime.  After  this,  the 
tanning  proper  consists  in  subjecting  the  skins  to  prolonged  immersion 
in  solutions  made  up  according  to  a  large  variety  of  formulae — the 
principle  of  all  of  which,  however,  seems  to  be  found  in  the  mixing  of 
various  organic  ingredients,  such  as  bran-mash,  oak-bark,  and  often 
dried  animal  excreta,  in  which  fermentation  and  acid  production  oc- 
cur. According  to  Haenlein,^  this  acidification  is  the  essential  by 
which  the  leather  is  sterilized  and  rendered  soft.  This  author  has 
described  the  Bacillus  corticalis,  which  he  found  in  fir-tree  bark  and 
to  which  he  ascribes  the  acid  fermentation  of  tanning  liquors  in  which 
this  ingredient  is  employed.  Wood,^  who  has  worked  extensively  upon 
the  subject,  has  attempted  to  substitute  pure  cultures  for  the  old  un- 
certain chance  mixtures  employed.  In  spite  of  these  investigations, 
however,  while  we  must  acknowledge  the  probable  importance  of  bac- 
teria in  the  tanning  process,  the  subject  is  by  no  means  on  a  scientific 
or  exact  basis. 

1  Alvarez,  Comptes  rend,  dc  I'acad.  des  sci.,  vol.  105. 

2  Haenlein,  Cent,  f .  Bakt.,  II,  i,  1895. 

»  Wood,  Jour.  Soc.  Chem.  Industry,  1895,  1899, 


SECTION  VI 

PATHOGENIC   PROTOZOA 

Frederick  F.  Russell,  M.D. 

INTRODUCTION 

In  the  practice  of  his  profession  the  physician  requires  a  knowl- 
edge of  the  pathogenic  protozoa  found  in  man  and  the  domestic  ani- 
mals and  of  their  closely  related  non-pathogenic  forms.  Quite  com- 
monly in  the  diagnosis  of  fevers  it  is  necessary  to  examine  the  blood 
of  the  same  patient  for  both  malaria  and  bacteria,  therefore  a  work- 
ing knowledge  of  the  principal  pathogenic  protozoa  is  essential.  In 
this  work  it  will  be  possible  to  describe  the  forms  only  of  medical 
interest,  and  the  reader  is  referred  to  other  works  for  further  infor- 
mation. 

The  protozoa  are  unicellular  animal  organisms  that  occur  singly  or 
in  temporary  colonies.  The  functions  of  the  animal  are  carried  out  by 
the  protoplasm  of  the  single  cell,  parts  of  which  may  be  differentiated 
for  special  purposes  and  are  then  called  organellae. 

CLASSIFICATION  OF  THE  PROTOZOA 

Class  I.  Sarcodina  (Bhizopoda) . — The  body  is  naked  or  encased  and 
the  animal  moves  by  means  of  protruding  temporary  processes  of 
the  body  called  pseudopods.  They  possess  one  or  many  nuclei 
and  reproduce  by  fission  or  multiplication  in  a  cyst. 
Order  I,  Amehce  (Lohosa). — Naked  or  with  a  simple" shell,  the  pseu- 
dopia  are  lobose  or  finger-shaped,  the  nucleus  is  usually  single  and 
there  is  sometimes  a  contractile  vacuole.    Example,  the  amehce. 

Class  II.  Mastigophora  (Flagellata). — They  possess  flagella  for 
locomotion  and  for  obtaining  food ;  they  may  be  naked  or  fur- 
nished with  a  membrane ;  many  forms  possess  nucleus,  contractile 
vacuole  and  a  small  groove  spoken  of  as  the  cytostome.  Ex- 
amples, the  trypanosomes  and  intestinal  flagellates. 

72J 


722  PATHOGENIC  PROTOZOA 

Class  III.  Sporozoa. — They  live  parasitically  in  the  tissues  o£  other 
animals,  ingesting  food  by  osmosis ;  they  have  no  cilia  in  the  adult 
stage  but  may  form  pseudopodia,  one  or  more  nuclei,  no  contractile 
vacuole,  reproduction  by  spores.  They  are  divided  into  two  sub- 
classes, telosporidia  and  neosporidia.  Examples,  gregarinida,  coc- 
cidiidea,  hemosporidia,  sarcosporidia,  etc. 

Class  IV.  Infusoria  (Ciliata), — The  body  is  generally  uniform  in 
shape,  with  cilia  and  contractile  vacuole,  and  usually  with  macro- 
and  micronucleus.    Examples,  parameciiim,  halantidium. 


CHAPTER   LV 
CLASS  I— SARCODINA   (RHIZOPODA) 

THE   AMEB-ffi 

These  organisms  belong  to  the  order  Amehina  (Ehrenberg).  They 
are  characterized  during  the  vegetative  stage  by  a  semifluid  consis- 
tence, permitting  rapid  changes  of  form,  ameboid  movements,  and 
progression  by  means  of  pseudopods.  There  is  no  internal  skeleton  and 
the  protoplasm  is  naked  and  may  be  differentiated  into  endo-  and 
ectoplasm,  and  in  some  cases  a  contractile  vacuole  is  present.  All 
forms  possess  one  or  more  nuclei.  Multiplication  takes  place  by  divi- 
sion into  two  or  more  daughter  cells.  Fertilization  takes  place  by  the 
conjugation  of  two  merozoites  and  possibly  by  autogamy. 

Since  some  flagellates  possess  an  ameboid  stage,  it  is  necessary  to 
know  most  of  the  life  cycle  of  an  organism  before  classifying  it  as  an 
ameba.  The  protoplasm  varies  greatly  in  its  consistency,  depending 
on  the  species  as  well  as  the  stage  of  the  life  cycle,  and  the  environ- 
ment and  food  supply.  Most  amebag,  including  all  the  parasitic  forms 
(endamebge),  possess  a  single  nucleus,  yet  Ameba  diploidea  and  Ameha 
hinucleata  always  have  two,  and  the  other  species  may  show  more. 
The  nucleus  of  all  types  possesses  a  karyosome.  The  nucleus  is  well 
developed  and  in  it  may  be  followed  either  a  simple  or  typical  mitosis. 
The  cytoplasm  is  usually  at  some  stage  divided  into  a  granular  endo- 
plasm  and  a  clear  or  hyaline  ectoplasm,  the  latter  forming  the  pseu- 
dopods by  which  the  animal  moves  from  place  to  place. 

Until  recent  years  all  ameboid  organisms  were  placed  in  the  genus 
Ameha,  but  Schaudinn  revived  a  genus  originated  by  Leidy  of  Phila- 
delphia, the  Endameha,  for  the  parasitic  species  which  have  many 
points  of  difference  from  the  free  living  varieties.  Of  the  free  living 
forms,  the  easiest  to  study  is  the  Ameha  proteus  (Pallas),  a  very  large 
organism,  200  microns  in  diameter,  found  frequently  in  stagnant 
water;  it,  however,  has  no  direct  importance  in  medicine.  Another 
group  of  free  living  amebae  is  of  some  interest,  because  of  the  con- 
fusion they  have  caused  in  the  study  of  parasitic  ameba ;  they  are  the 
so-called  **limax  amebae,*'  which  have  been  cultivated  on  agar,  and 
for  this  genus  Chatton  (1912)  has  proposed  the  name  Vahlkampfia. 
They  are  small  organisms,  5  to  30  microns  in  diameter,  provided  with 

723 


724 


PATHOGENIC  PROTOZOA 


finger  like  or  spinous  pseudopodia,  and  characterized  by  a  nucleus  with 
a  large  karyosome  and  a  single  nucleated  resistance  cyst  in  which  no 
multiplication  occurs.  They  have  repeatedly  been  cultivated  from  hu- 
man dysenteric  stools,  from  the  air,  and  apparently  from  liver  abscess 
pus.  It  has  been  shown,  beyond  doubt,  that  they  are  harmless  to  man, 
and  tha't  they  pass  through  the  intestinal  tract  with  food  and  water 
in  the  cyst  form.  While  they  will  develop  in  cultures  at  body  tempera- 
ture, a  better  growth  is  obtained  at  the  temperature  of  the  room.    Since 


f 

% 

0 
r            *• 

• 

Fig.  156. — Endameba  histolytica.      Vegetative  form  showing  histolytica  type  of 
nucleus.     (Army  Medical  School  Collection,  Washington,  D.  C.) 

the  true  parasitic  amebae  have  never  been  cultivated  on  artificial  media, 
the  Vahlkampfia  may  be  dismissed  with  the  statement  that  they  are 
not  pathogenic. 

Leidy  's  genus  Endameba  includes  all  parasitic  forms,  and  is  char- 
acterized, among  other  things,  by  the  absence  of  a  contractile  vacuole, 
which  is  always  present  in  Ameba  and  Vahlkampfia.  It  was  proposed 
for  the  large  ameba  parasitic  in  the  cockroach,  Endameba  hlattce,  and 
the  genus  is  a  natural  one  and  well  established.  The  species  of  impor- 
tance to  physicians  are  Endameba  histolytica,  Endameba  coli  and 
Endameba  gingivalis. 


SARCODINA 


725 


ENDAMEBA  HISTOLYTICA 

(Endameha  tetragena   [Viereck],  Endameha  africana    [Hartmann], 

Endameha  nipponica  [Koidzumi,  pro  parte],  Endameha 

tropicalis  [Lesage,  pro  parte] ) 

Amebae  as  a  cause  of  disease  were  first  described  by  Lambl  of 
Prague,  in  1860,  who  found  them  present  in  the  stools  from  a  case 
of  severe  diarrhea  in  a  child.  In  1870  Lewis  and  Cunningham  found 
amebae  in  20  per  cent  of  the  stools  of  cholera  patients,  but  attached  no 
pathogenic  importance  to  them.  The  first  accurate  description  we  owe 
to  Loesch  of  Petrograd,  who  in  1875  studied  an  undoubted  case  of 


i'  IG.  157. — Endameba  histolytica.     Vegetative  form,  simple  division.     Tetragena 
type  of  nucleus.    (X  1300.)    (Army  Medical  School  Collection,  Washington,  D.  C.) 


amebic  dysentery  with  relapses,  and  he  named  the  organism  Ameha 
coll.  He  was  further  successful  in  reproducing  the  disease  in  a  dog, 
and  thus  began  its  experimental  investigation.  Not  much  progress 
was  made  until  Kartulis  in  Egypt  began,  in  1886,  the  publication  of 
a  long  series  of  studies  which  has  continued  up  to  the  present  time, 
and  because  of  the  rich  clinical  and  pathological  material  at  his  dis- 
posal his  work  has  been  of  the  greatest  value.  In  1890  Osier  published 
the  first  paper  in  America.  He  was  followed  by  Musser  and  Stengel 
and,  in  1891,  by  Dock,  and  Councilman  and  Lafleur.     The  work  of  the 


726  PATHOGENIC  PROTOZOA 

last  two  authors  was  especially  complete  and  firmly  established  the 
entity  of  this  disease  in  America.  In  1902  Jiirgens  differentiated  the 
pathogenic  ameba  from  the  harmless,  and  in  1903  the  epoch-making 
work  of  Schaudinn  appeared.  This  author,  who  was  a  zoologist  by 
training,  showed  clearly  that  there  were  two  forms  of  parasitic  amebse 
and  he  followed  out  most  of  the  details  in  their  life  history,  renaming 
them  Endameha  histolytica  and  Endameba  coli.  Schaudinn 's  work 
has  been  generally  confirmed,  in  this  country  by  Craig,  Whitmore, 
Walker,  Darling  and  others. 

CLINICAL   DYSENTERY 

Dysentery  as  a  disease  has  been  known  from  the  earliest  times  and 
references  are  found  to  it  in  Sanscrit  and  Egyptian  literature  and  in 
early  Greek  and  Roman  writings.  Until  recent  years  its  etiology  was 
obscure,  but  we  now  recognize  two  separate  forms,  bacillary  and 
amebic;  the  former  has  already  been  described  under  the  dysentery 
bacilli.  Amebic  dysentery  is  a  distinct  clinical  entity,  and  runs  a 
course  quite  different  from  the  bacillary  form.  It  begins  gradually, 
and  in  some  cases  is  chronic  in  character  from  the  start.  Usually  there 
is  no  rise  in  temperature  nor  any  great  change  in  weight  or  health 
until  the  disease  has  existed  some  time.  The  bowel  movements  become 
gradually  more  frequent  and  the  fecal  matter  is  accompanied  by 
larger  and  larger  amounts  of  mucus  and  blood.  As  the  disease  pro- 
gresses and  more  and  more  of  the  colon  is  involved  the  amount  of  blood 
and  mucus  increases  until  the  stool  contains  little  else.  The  colicky 
pains  increase  in  frequency  and  severity  and  there  is  added  tenesmus 
and  finally  nausea  and  vomiting.  The  patient  loses  flesh  and  strength 
and  when  the  stools  increase  to  20  and  30  daily,  becomes  bed-ridden. 
The  abdomen  is  concave  and  tender  on  pressure,  especially  over  the 
colon.  The  course  of  the  disease,  if  untreated,  tends  to  progress  with 
periods  of  remission,  and  spontaneous  cure  probably  does  not  occur. 
Bacillary  dysentery,  it  will  be  remembered,  is  a  disease  with  a  short  in- 
cubation period  and  an  acute  onset ;  after  two  or  three  days '  illness  the 
bacillary  case  is  confined  to  bed,  is  pale,  weak,  emaciated  and  presents 
every  evidence  of  profound  toxemia;  an  amebic  case,  sick  the  same 
length  of  time,  will  be  up  and  about  and  perhaps  will  not  have  applied 
for  treatment. 

Complications. — A  common  and  most  dangerous  complication  is 
abscess  of  the  liver.    The  amebae  travel  from  the  ulcers  in  the  colon  by 


SARCODINA  727 

way  of  the  lymphatics  to  the  liver  and  there  set  up  a  liquefying  ne- 
crosis of  the  parenchyma.  The  liquefied  portion  contains  a  reddish  or 
chocolate-colored  fluid,  which  is  not  pus  in  the  ordinary  sense,  althouj>h 
it  may  become  a  pus-containing  abscess  if  secondary  bacterial  infection 
occurs.  Liver  abscesses  may  be  single,  but  are  much  more  often  mul- 
tiple, and  at  times  the  whole  liver  may  be  riddled  with  large  and  small 
abscess  cavities;  both  right  and  left  lobes  may  be  involved.  If  sur- 
gical interference  be  withheld,  the  abscess  increases  in  size,  approaches 
the  surface,  and  finally  ruptures  into  the  lung  through  the  diaphragm 
or  into  the  peritoneal  cavity. 

At  autopsy  the  lesions  are  found  in  the  colon,  principally  at  the 
sigmoid  flexure  and  in  the  cecum,  though  in  chronic  cases  the  whole 
colon  is  involved,  showing  ulcers  with  undermined  edges,  swollen  soli- 
tary follicles  and  a  hemorrhagic-catarrhal  inflammation  of  the  mucous 
membrane.  The  ulcers,  readily  differentiated  from  those  caused  by 
the  tubercle  bacillus,  are  of  all  sizes,  shallow  or  deep,  and  are  charac- 
terized by  irregular  margins  and  undermined  edges.  Fresh  smears 
made  at  autopsy  will  show  vegetative  amebae.  In  chronic  cases,  the 
colon  is  a  mass  of  scars  and  ulcers  and  acutely  inflamed,  swollen  and 
thickened  mucous  membrane  resting  on  a  hypertrophied  submucosa. 
The  severe  and  chronic  forms  of  the  disease  are  now  as  rare  as  they 
were  formerly  common  as  a  result  of  the  present  specific  treatment 
with  emetin. 

Geographical  Distribution. — Although  amebic  dysentery  is  classed 
among  the  tropical  diseases,  it  is  by  no  means  confined  to  the  tropics. 
In  the  United  States,  for  example,  it  is  endemic  as  far  north  as  Balti- 
more and  Washington,  and  cases  are  not  very  infrequent  in  the  north- 
ern tier  of  states;  hence  one  must  examine  the  stools  for  amebae  in 
dysenteric  cases  regardless  of  the  location  of  the  patient's  home. 

Diagnosis. — While  the  history  of  the  case  may  suggest  amebic  infec- 
tion, the  diagnosis  can  onTy  be  made  with  certainty  by  microscopic 
examination  of  the  stool.  For  this  purpose  the  examination  should  be 
made  as  soon  after  the  stool  is  passed  as  possible;  and  in  this  disease 
it  is  usually  practicable  to  have  the  patient  come  to  the  hospital,  clinic 
or  office  and  pass  a  stool  there.  It  may  then  be  examined  imme- 
diately. If  this  is  impracticable,  the  stool  may  be  kept  warm  and  sent 
to  the  laboratory  in  a  small  glass  jar  inside  a  tin  pail  partly  filled 
with  water  at  body  heat;  a  little  cloth  or  absorbent  cotton  will  hold 
the  hot  water  and  prevent  splashing  during  transit.  The  stool  will 
show  bloody  mucous  masses,  and  small  drops  of  this  are  placed  on 


728  PATHOGENIC  PROTOZOA 

slides,  covered  with  a  glass  and  ringed  with  warm  vaseline  to  prevent 
evaporation.  The  preparation,  to  be  of  value,  must  be  thin,  and  the 
bloody  mucus  may  be  diluted  with  salt  solution  if  necessary.  Except 
in  hot  weather,  the  slide  should  be  examined  on  a  warm  stage,  or  the 
slide  may  be  warmed  by  placing  heated  coins  on  it,  near  the  cover 
glass.  At  least  half  a  dozen  slides  should  be  examined  before  reporting 
a  negative  result. 

Stained  preparations  are  not  difficult  to  prepare,  although  the 
process  requires  some  time  and  care.  As  in  most  zoological  work,  wet, 
rather  than  dry,  fixation  is  used.  Thin  smears  are  made  on  cover 
glasses  or  slides  and  before  they  can  dry  are  covered  with  or  immersed 
in  Schaudinn's  fluid.  This  is  a  mixture  of  two  parts  of  a  saturated 
solution  of  bichlorid  of  mercury  in  normal  salt  solution  and  one  part 
of  absolute  alcohol.  The  mercuric  solution  is  prepared  by  adding  to 
boiling  normal  salt  solution  a  little  more  mercury  than  will  dissolve;' 
on  cooling,  some  of  the  bichlorid  crystallizes  out.  At  no  stage  of  the 
process  must  the  preparation  become  dry  or  the  smear  is  worthless. 

1.  Fix  in  hot  (60°  C.)  Schaudinn's  fluid,  5  to  10  minutes. 

2.  Harden  in  70  per  cent  alcohol  10  to  30  minutes,  then  wash  in 
70  per  cent  alcohol  to  which  a  few  drops  of  tincture  of  iodin  have  been 
added  until  it  is  distinctly  colored — 10  minutes ;  store  in  70  or  80  per 
cent  alcohol  until  ready  to  stain. 

3.  The  Rosenbusch  hematoxylin  is  quite  satisfactory.  Transfer  the 
slides  to  distilled  water  and  change  several  times  until  they  are  free 
from  alcohol,  then  immerse  in  3.5  per  cent  iron-alum  solution  for  from 
half  an  hour  to  over  night. 

4.  Stain  in  the  following  solution,  after  rapid  washing  in  distilled 
water: 

(a)   1  per  cent  hematoxylin  in  95  per  cent  alcohol. 

(&)   Saturated  aqueous  solution  of  lithium  carbonate. 

Solution  (h)  is  added  to  solution  (a)  until  the  mixture  is  a 
cherry  red,  four  or  five  drops  of  lithium  to  10  c.c.  of  hematoxylin 
is  sufficient. 

The  solution  is  either  pipetted  onto  the  slides  or  .they  are  im- 
mersed in  it.     Stain  from  20  minutes  to  over  night. 

5.  Wash  thoroughly  in  distilled  water. 

6.  Differentiate  with  a  weak  iron-alum  solution  (three  parts  of 
distilled  water  to  one  of  the  iron-alum  solution  is  satisfactory),  until 


SAHCODINA 


729 


the  slide  under  the  microscope  shows  the  structure  of  the  nucleus ;  the 
examination  is  made  in  water  under  a  cover  glass. 

7.  When  the  differentiation  is  complete  the  slide  is  washed  in  dis- 
tilled water  and  passed  through  graded  alcohols,  80,  95  and  absolute 
into  xylol  and  xylol-balsatm.    This  stain  is  permanent. 

Romanowski  stains  on  dried  smears  may  be  used,  but  are  not  so 
good. 

In  fresh  specimens  Endameba  histolytica  presents  the  following^ 
appearance:  the  vegetative  forms  are  pale,  unstained  with  bile,  and 
are  seen  to  be  large  bodies,  20  to  30  microns  in  diameter,  consisting  of 
endo-  and  ectoplasm,  and  often  showing  a  delicate  nucleus  and  also 


Fig.  158. — Endameba  histolytica.     (Army  Medical  School  Collection, 
Washington,  D.  C.) 

many  inclusions  in  the  digestive  vacuoles,  principall}^  red  blood  cells. 
The  organisms  for  several  hours  after  the  stool  is  passed  remain  ac- 
tively motile,  pushing  out  clear,  glass-like  pseudopods,  into  which  the 
granular  endoplasm  pours  as  the  ameba  progresses  across  the  field. 
Even  when  there  is  no  progression  the  pseudopods  are  protruded  or 
retracted  first  in  one  then  in  another  direction.  There  is  always,  dur- 
ing motion,  a  distinct  separation  of  the  clear  ectoplasm  from  the 
granular  endoplasm,  and  the  latter,  in  acute  cases  especially,  contains 
many  red  blood  cells,  occasional  examples  showing  as  many  as  twenty 
or  thirty.  The  presence  of  red  blood  cells  either  entire  or  partly  di- 
gested is  characteristic  of  Endameba  histolytica,  since  they  are  pres- 
ent in  Endameba  coli  in  only  rare  instances.  The  ameba  is  sometimes 
greenish,  and  it  is  supposed  that  this  color  is  due  to  hemaglobin 
liberated  from  the  ingested  red  cells.    The  pseudopods  of  this  species 


730 


PATHOGENIC  PROTOZOA 


are  clear,  glassy  and  evidently  viscid  and  dense  and  have  given  it  its 
name  ''histolytica,"  since  Schaudinn  states  that  he  saw  the  ameba 
penetrate  the  mucous  membrane,  the  pseudopods  dissecting  apart  the 
epithelial  cells.  The  nucleus,  when  the  endoplasm  is  packed  with  in- 
clusions, may  not  be  visible,  but  further  search  will  reveal  amebse 
showing  a  nucleus.  It  is  vesicular,  with  a  delicate  limiting  membrane,, 
and  as  it  is  highly  refractile,  may  appear  as  a  clear  bright  spot.  As 
the  specimen  grows  older  the  amebse  lose  much  of  their  motility  and 
the  nucleus  may  become  clearly  visible,  revealing  small  chromatic  dots 
or  masses  adherent  to  its  inner  surface  and  a  small  central  karyosome. 


Fig.  159.  —  End  ameba  histolytica. 
(Army  Med.  School  Collection,  Wash- 
ington, D.  C.) 


Fig.  160.  —  End  ameba  histolytica 
(X  1150.)  (Army  Med.  School 
Collection,  Washington,  D.  C.) 


The  cysts  are  round,  quite  small,  about  10  microns,  and  show  four 
small  ring-like  nuclei ;  the  wall  is  distinct  and  often  double-contoured. 

The  motile  amebae  cannot  be  confused  with  anything  else,  but  when 
in  the  resting  stage  they  have  been  mistaken  for  swollen  and  edematous 
epithelial  cells.  A  little  attention  to  the  nucleus  will  prevent  this 
error,  since  the  tissue-cell  nucleus  is  large,  distinct,  and  entirely  dif- 
ferent from  the  nucleus  of  an  ameba. 

In  specimens  stained  with  hematoxylin  the  finer  details,  especially 
in  the  nucleus,  may  be  studied,  but  stained  preparations  are  never  nec- 
essary for  clinical  diagnosis.  In  smears  from  fresh  cases  vegetative 
forms  only  are  found,  later  many  degenerative  forms  appear  and  dur- 
ing convalescence  only  cysts  may  be  seen.    In  stained  specimens  there 


SARCODINA  731 

is  rarely  any  separation  of  ectoplasm  and  endoplasm,  but  the  nucleus 
is  always  visible.  The  cytoplasm  is  granular  and  has  a  coarse  honey- 
combed" appearance.  The  nucleus  shows  a  distinct,  though  delicate, 
limiting  membrane,  on  the  inner  surface  of  which  are  few  or  many 
chromatin  dots.  In  the  center  is  a  small  karyosome,  which  may  show 
a  central  body  or  centriole.  The  outer  zone  of  the  nucleus  has  a  honey- 
comb structure,  in  which  are  imbedded  granules  of  chromatin. 

Multiplication  in  the  vegetative  stage  is  by  division  into  two  daugh- 
ter cells,  and  dividing  cells  are  common  after  the  disease  has  existed 
for  some  time  and  the  amebge  are  preparing  to  encyst.  At  such  time's, 
due  probably  to  rapid  division,  the  amebae  are  small  and  the  nucleus 
shows  various  stages  of  a  simple  mitosis;  at  the  poles  of  the  delicate 
spindle  may  be  seen  the  two  new  centrioles ;  this  may  be  detected  even 
in  unstained  specimens.  ^ 

Degenerative  Forms. — These  are  extremely  common  in  stale  stools, 
in  cases  during  convalescence,  or  under  active  treatment,  and  also  in 
experimental  dysentery  in  the  cat,  and  they  have  led  to  much  con- 
fusion in  the  past.  The  nucleus  breaks  up  into  fragments  and  chro- 
matin masses  are  extruded  into  the  cytoplasm  in  irregular  forms,  and 
parts  of  the  cells  are  apparently  budded  off.  At  one  time  the  budding 
process  was  looked  upon  as  normal  by  Schaudinn  and  his  followers, 
but  there  is  now  little  doubt  JJiat  both  spores  and  buds  are  degenera- 
tive changes  and  that  the  animal  multiplies  only  by  binary  fission  in 
the  vegetative  forms  or  by  the  development  of  four  nuclei  in  the  cysts. 

Cyst  Formation. — The  cyst  of  Endameba  histolytica  w^as  first  de- 
scribed by  Viereck  as  Endameha  tetragena,  and  for  a  time  was  believed 
to  be  a  new  species.  In  fact,  Hartmann  described  a  vegetative  stage 
of  Endameba  tetragena  as  Endameha  africana,  afterwards  accepting 
the  name  "  tetragena, ' '  but  it  is  now  apparent  that  tetragena  is  merely 
the  end,  or  cyst  stage,  of  Endameba  histolytica,  which  had  formerly 
been  overlooked.  Cysts  are  not  easily  found  in  all  cases,  and  it  is 
possible  that  when  treatment  is  vigorous  they  never  develop.  They 
are,  without  doubt,  the  form  in  which  the  parasite  leaves  the  body  to 
infect  new  victims ;  because  of  their  heavy  cyst  wall  they  are  quite  re- 
sistant to  drying  and  other  harmful  influences.  It  is  also  possible  that 
they  may  be  retained  in  the  wall  of  the  colon  and  so  be  the  starting- 
point  of  the  relapse,  which  is  so  characteristic  a  feature  of  amebic 
dysentery.  The  protoplasm  of  the  cyst  and  the  precystic  stage  is 
granular,  but  shows  no  vacuoles  nor  cell  inclusions.  The  nucleus  un- 
dergoes division  by  mitosis  first  into  two,  and  then  four  small  ring-like 


732 


PATHOGENIC  PROTOZOA 


4 


iff"  ^j. 


nuclei,  and  the  presence  of  these  four  nucleated  cysts  is  pathognomonic 
of  the  disease.  They  may  be  found  most  abundantly,  not  in  the  small 
amount  of  mucus  which  may  adhere  to  the  formed  feces,  but  in  surface 

scrapings  from  the  fe- 

Pcal  mass.  In  addition 
to  the  four  small  ring- 
^  like  nuclei,  the  cysts 
contain  few  or  many 
clumps  of  chromatin; 
these  in  total  mass  may 
be  many  times  greater 
than  the  nucleus,  and 
it  is  impossible,  there- 
fore, that  they  are 
simply  extruded  from 
the  nucleus;  evidently, 
the  chromatin  grains, 
while  in  the  cytoplasm, 
increase  in  size  and 
number.  In  hematoxy- 
lin stains  no  structure 
in  these  masses  is  dis- 
cernible and  their  func- 
tion is  unknown;  after 
a  time  they  disappear 
and  one  finds  cysts  quite  free  of  them.  The  presence,  however,  of 
many  large  chromatin  masses  in  the  cysts  is  characteristic  of  Enda- 
meba  histolytica. 

Fertilization  inside  the  cyst  has  not  been  demonstrated,  but  it  is 
possible  that  the  four  young  amebae,  liberated  from  the  cyst  when  in- 
gested by  a  new  host,  are  gamets,  and  that  conjugation  takes  place 
between  them,  as  in  the  case  with  Endameba  blattae. 

The  treatment  of  amebic  dysentery,  to  be  effective,  must  be  radical 
and  persistent,  and  may  be  compared  to  the  treatment  of  malaria  with 
quinine.  For  many  years  the  English  in  India,  with  a  few  followers 
in  other  parts  of  the  world,  had  treated  dysentery  with  ipecac  in  mas- 
sive doses,  with  wonderful  results  in  some  cases  and  failure  in  others. 
The  treatment  was  quite  disagreeable  and  not  entirely  satisfactory. 
Vedder,  in  1911,  examined  the  various  alkaloids  of  ipecac  and  found 
that  emetin  was  strongly  amebacidal,  and  he  recommended  its  use  for 


0, 

m 

i 

i 


Fig. 


161. — Endameba  coli.      (Army    Med. 
Collection,  Washington,  D.  C.) 


SARCODINA 


733 


this  disease.  Rogers,  in  India,  following  out  this  suggestion,  soon  re- 
ported excellent  results,  and  the  drug  is  now  accepted  as  a  specific. 
It  is  administered  hypodermically  in  ^-grain  doses  three  times  a  day 
at  first,  then  twice  and  later  once  daily  until  a  total  of  ten  grains  has 
been  administered  (Vedder).  In  addition,  the  patient  is  put  to  bed 
and  placed  on  a  milk  diet.  During  convalescence  large  doses  of  bis- 
muth subnitrate,  a  heaping  teaspoonful  suspended  in  water  or  milk, 
may  be  given  (Eieeks).  Relapses  may  be  prevented  by  occasional 
examination  of  the  stools  for  cysts  and  a  course  of  emetin  if  they  are 


Fig.  162. — ^Endameba  coli. 


Typical  nucleus.     (Army  Med.  School  Collection, 
Washington,  D.  C.) 


found.  As  a  result  of  the  emetin  treatment  and  exact  diagnosis  the 
clinical  picture  of  amebic  dysentery  has  completely  changed,  and  we 
no  longer  see  the  weak  and  emaciated  dysenteries  who  formerly 
crowded  the  wards  of  tropical  hospitals. 

Prevention. — One  significant  fact  appears  in  the  epidemiology  of 
the  disease — ^it  always  occurs  sporadically  and  never  in  explosive  epi- 
demics such  as  we  see  in  water-borne  diseases,  like  typhoid  and  chol- 
era; house  epidemics  are,  on  the  contrary,  not  uncommon.     This  fact 


734 


PATHOGENIC  PROTOZOA 


points  to  the  importance  of  contact,  and  perhaps  flies,  as  the  chief 
agents  in  its  spread.  Extreme  cleanliness  among  the  servants  and  in 
the  kitchen  will  prevent  the  transfer  of  histolytica  cysts  from  the  ill 
to  the  well.  The  disease  has  disappeared  from  the  Panama  Canal 
Zone,  where  it  formerly  was  common,  since  the  introduction  of  good 
water  and  sewer  systems  and  better  hygienic  conditions. 

ENDAMEBA  COLI  (Loesch  emend.  Schaudinn) 

{Endameha  nipponica  [Koidzumi,  pro  parte]  and  possibly 
Endameha  minuta  [Elmassian]) 

This  is  a  harmless  parasite  of  man,  and  its  presence  in  stools,  at 
one  time,  gave  rise  to  much  confusion,  and  in  the  minds  of  many, 

threw  doubt  upon  the  ex- 
istence of  a  form  of  dys- 
entery due  to  ameba,  since 
it  was  found  not  infre- 
quently in  healthy  individ- 
uals. Schaudinn  found  it 
present  in  the  stools  of 
fifty  per  cent  of  the  per- 
sons examined  in  East 
Prussia,  in  Berlin  in 
twenty  per  cent,  and  in 
Istria  in  sixty  per  cent. 
Craig,  and  Craig  and  Ash- 
burn  found  it  present  in 
176,  or  fifty-eight  per  cent, 
of  307  examinations  of 
healthy  American  soldiers. 
Craig  was  able  to  follow* 
some  individuals  for  four 
to  six  years,  during  which 
time  they  constantly 
showed  Endameha  coli  in  the  feces,  yet  never  developed  dysentery. 
The  organism  seems  to  be  found  in  all  countries,  regardless  of  climate. 
Its  recognition  and  separation  from  histolytica  we  owe  to  Schaudinn, 
Jiirgens,  Craig  and  others. 

In  size  it  varies  from  ten  to  forty  microns,  the  average  being  be- 
tween twenty  and  forty.  The  ectoplasm  is  never  seen  except  during 
movement,  and  it  is  then  hyaline,  and  only  slightly  refractile,  and 


Yid.  1G3.^Endameba  coli.  Small  precystic 
form.  (Army  Med.  School  Collection,  Wash- 
ington, D.  C.) 


SARCODINA  735 

much  more  fluid  than  in  histolytica.  The  digestive  vacuoles  rarely 
contain  red  blood  cells,  but  are  filled  with  cocci  and  bacilli,  a  form  of 
food  rarely  seen  in  histolytica.  In  general,  the  vacuoles  are  larger 
and  more  numerous  in  coli  than  in  histolytica,  and  the  motility  is 
feebler.  In  fresh  specimens  the  nucleus  is  rather  easier  to  find  than 
in  histolytica,  and  is  distinctly  outlined  by  a  heavy,  double-contoured 
membrane.  The  nucleus,  as  in  all  ameba,  is  vesicular,  and  shows  a 
small  karyosome  and  dots  of  chromatin  on  the  nuclear  membrane  and 
imbedded  in  the  nuclear  network. 


Fig.  164. — ^Endameba  coli  Cyst.     (Army  Med.  School  Collection, 
Washington,  D.  C.) 

Multiplication  in  the  vegetative  stage  is- by  binary  fission  of  the 
nucleus  and  the  cytoplasm,  resulting  in  two  daughter  cells,  or  the 
nucleus  may  continue  to  divide  into  four  or  eight  daughter  nuclei 
before  the  cytoplasmic  division  begins,  producing,  in  the  end,  two, 
four,  or  eight  daughter  cells. 

Cyst  Formation. — This  is  characteristic  of  the  species,  and  it  fur- 
nished one  of  the  principal  reasons  for  Schaudinn  's  separation  of  coli 
and  histolytica.     Before  encysting  the  animal  frees  itself  of  all  in- 


736  PATHOGENIC  PROTOZOA 

elusions  and  becomes  clear,  transparent,  and  assumes  a  spherical  form, 
and  secretes  a  cyst  wall.  The  nucleus  divides  first  into  two,  then  four, 
and  finally  eight  daughter  nuclei;  there  is  usually  a  large  vacuole 
found  in  the  cyst  during  this  division,  but  its  function  is  uncertain. 
Schaudinn  described  a  complicated  autogamy  in  the  cyst,  yet  later 
researches  by  Hartmann  and  Whitmore  show  nothing  more  than  re- 
peated binary  division  of  the  nucleus.  The  normal  number  of  nuclei 
in  a  coli  cyst  is  eight,  yet  occasionally  cysts  are  seen  in  which  division 
has  gone  on  until  there  are  as  many  as  sixteen. 

Cats  or  human  beings  may  be  parasitized  by  feeding  material  con- 
taining coli  cysts,  and  in  nature,  as  the  cysts  are  the  resistant  forms  of 
the  parasite,  the  infection  is  probably  transmitted  from  one  host  to 

another  by  means  of  them.     No  disease, 
however,  results,  though  the   amebae  con- 
tinue to  be  present  in  the  stools  for  years. 
It  is  possible  that  fertilization-takes  place 
J      between    the    young    amebae     (gametes?), 
which  are  liberated  when  the  cyst  dissolves 
,      in  a  new  host,  as  is  the  case  with  Enda- 
meba  blattse. 


-^_--'  ENDAMEBA  GINGIVALIS 

Fig.  165.-ENDAMEBA  coli.        (^^^^  1849,  emend,  von  Prowazek  1904) 
Cyst  showing  eight  nu-  i        .       n         -,     •       ,,        , 

clei.  (Arch,  fiir  Protisten-  Ihis    ameba    is    lound    m    the    human 

kunde,  1912,  xxiv.)  mouth  both  in  health  and  disease.     It  has 

been  described  at  different  times  under 
various  names  {huccalis,  dent  alls)  by  Gros,  Steinberg,  von  Prowazek, 
Lewald,  Smith  and  Barrett,  Chiavaro  and  Craig,  and  quite  recently 
has  been  suggested  as  the  cause  of  pyorrhea  alveolaris  by  Smith  and 
Barrett  and  Bass  and  Johns.  It  is  widely  distributed,  and  has  been 
reported  from  all  quarters  of  the  world. 

The  organism  is  easily  found  in  the  tartar  at  the  base  of  the  teeth, 
in  cavities  in  the  teeth,  and  even  at  the  gum  margin  in  healthy  mouths. 
It  varies  in  size  from  seven  to  thirty-five  microns,  averaging  between 
twelve  and  twenty  (Craig).  Motility  is  well  marked,  though  it  is  not 
so  active  an  organism  as  histolytica,  the  pseudopods,  mostly  short  and 
blunt,  being  formed  of  the  clear,  slightly  refractile  ectoplasm.  The 
endoplasm  is  granular,  contains  the  nucleus  and  many  food  vacuoles 
containing  nuclei  of  leucocytes  and  granular  matter,  and  rarely  a  few 


SARCODINA  737 

red  blood  cells.  The  nucleus  is  small,  and  in  fresh  specimens  is  usually 
invisible. 

Before  encysting  the  parasites  are  much  reduced  in  size,  and  the 
cytoplasm  frees  itself  from  all  inclusions  and  becomes  clear,  spherical 
and  immobile.  The  cysts  are  small,  eight  to  ten  microns,  circular  and 
definitely  outlined,  sometimes  with  a  double  contour,  and  in  stained 
specimens  the  nucleus  is  always  visible.  It  is  small,  averaging  only 
three  microns  (Craig),  making  it  smaller  than  in  histolytica  and  coli. 
The  limiting  membrane,  whil-e  not  heavy,  is  distinct,  and  encloses  a 
nuclear  body  having  very  little' chromatin  other  than  the  small  cen- 
trally located  karyosome.  Multiplication  occurs  only  in  the  vegetative 
state  and  by  binary  fission. 

Cyst  Formation. — Cysts  are  rarely  observed,  and  then  in  small  num- 
bers ;  the  cyst  wall  is  not  heavy,  but  may  show  a  double  contour ;  the 
protoplasm  is  clear,  free  from  all  inclusions  and  vacuoles  and  shows  a 
single  small  nucleus,  but  without  any  signs  of  multiplication.  It  is 
apparent,  therefore,  that  the  cyst  is  a  protective  stage  and  has  nothing 
to  do  with  reproduction,  which  occurs  in  the  vegetative  state  only.  In 
this  respect  it  resembles  the  Vcthlkampfia. 

Although  the  organism  is  almost  constantly  present  in  pyorrhea 
alveolaris  it  is  also  found  in  healthy  mouths,  and  in  the  absence  of  all 
experimental  proof,  it  is  doubtful  if  the  organism  is  of  pathological 
importance.  Emetin  has  a  decided  effect  upon  many  cases  of  pyorrhea 
alveolaris,  and  under  that  treatment  alone  the  disease  may  disappear; 
the  nature  of  its  therapeutic  action  is  not  yet  clear,  and  does  not  neces- 
sarily indicate  any  etiological  relationship. 


CHAPTER   LVI 

CLASS  II— MASTIGOPHORA   (DIESING) 
SUB-CLASS— FLAGELLATA   (COHN   EMEND.   BtJTSCHLI) 


ORDER  I— POLYMASTIGINA  (Blochmann) 

These  are  flagellates,  possessing  three  to  eight  flagella. 

Genus  1.  —  Trichomonas  (Donne,  1837).  —  These  have  pyroform 

(pear-shaped)  bodies,  rounded  in  front  and  tapering  to  a  point  behind, 

provided  with  three  long  flagella,  often  matted  together  at  the  anterior 

end.    An  internal  supporting  structure,  known  as  the  axial  filament  or 

axostyle,  is  present.  There  is  an  undulat- 
ing membrane  bordered  by  a  trailing 
flagellum  that  begins  anteriorly  and  runs 
obliquely  backwards. 

Trichomonas  vaginalis  (Donne). — The 
organism  is  fifteen  to  twenty-five  microns 
long  and  seven  to  twelve  wide;  it  is  pro- 
vided with  three  flagella  and  an  undulat- 
ing membrane.  It  is  found  in  the  vaginal 
secretion  only  when  it  is  acid,  and  in  three 
instances  it  has  been  transmitted  to  the 
male. 

Trichomonas  intestinalis  (R.  Leuckard, 
1879). — This  parasite  is  practically  indis- 
tinguishable from  Ttichomonas  vaginalis. 
It  occurs  in  the  small  intestine  and  appears 
in  the  stools  during  diarrheal  attacks,  but 
is  probably  non-pathogenic.  It  is  readily 
found  in  the  intestine  and  colon  of  mice  and 
guinea-pigs.  In  fresh  specimens  (protected 
with  a  cover  glass  and  vaseline)  it  is  ac- 
tively motile,  but  the  undulating  membrane  is  difficult  to  detect  until 
the  movement  has  slowed  down. 

738 


Fig.  166. — Trichomonas  in- 
TESTiNALis.  (After 
Brumpt,  "  Precis  de  Para- 
sitologie,"  1914  ed.) 


MASTIGOPHORA  739 

The  presence  of  cystic  forms  has  been  questioned,  and  two  quite 
different  forms  have  been  called  resistance  or  dauer  cysts.  The  earlier 
one,  described  by  Ucke  (1908),  Bohne  and  von  Prowazek  (1908),  and 
Benson  (1910),  is  a  fairly  large  body^  showing  a  double  contour  and  a 
central  homogeneous  mass,  perhaps  food  material,  and  an  outer  ring- 
like body  containing  two  or  more  nuclei.  Brumpt  and  Alexieff  believe 
this  form  to  be  a  fungus,  having  no  relation  to  the  trichomonad,  and 
have  called  it  ' '  Blastocystis  hominis."  Lynch  ^  agrees  with  these  au- 
thors, and  describes  an  altogether  different  body  as  the  resistant  form. 
It  is  six  by  eight  microns  in  size  and  perfectly  symmetrical  in  shape. 
The  wall  is  distinct,  and  there  is  a  clear  space  between  it  and  the  body 
of  the  parasite.  The  nucleus,  undulating  membrane  and  flagella  re- 
main visible  in  the  cyst,  but  Lynch  was  unable  to  detect  any  change 
in  the  parasite  indicating  intracystic  multiplication. 

Infection  takes  place  probably  by  contact,  and,  as  in  typhoid  fever, 
food,  fingers  and  flies  carry  the  resistant  forms  from  one  individual  to 
another.  Among  the  natives  of  tropical  countries  infection  is  almost 
universal,  but  the  parasites  are  rarely  seen  in  the  large  cities  of  the 
North. 

Genus  2. — Tetramitis  mesnili  (Wenyon,  1910). — Macrostoma  mes- 
nili,  Chilomastix  mesnili,  Fanapapea  intestinalis.  This  organism,  first 
described  by  Wenyon,  from  a  native  of  the  Bahamas,  differs  from  tri- 
chomonas by  the  possession  of  a  deep  groove 
or  cystotome,  in  which  is  found  the  undulat- 
ing membrane.  It  is  present  in  diarrheal  dis- 
charges, but  its  pathogenicity  is  doubtful. 


Fig.  167. — Lamblia  intestinalis.    Cyst  formation.    (After  Doflein,  "Lehrbuchder 

Protozoenkunde. ' ' ) 

Genus  3. — Lamblia  intestinalis   (Lambl,  1859). — The  lambia  are 
peculiar,   bilaterally   symmetrical,   pear-shaped    organisms,    provided 

1  Lynch,  Kenneth  M.,  Jour.  Parasitol.,  Urbana,  1916,  iii,  28. 


740  PATHOGENIC  PROTOZOA 

with  a  sucking  disk  anteriorly.  There  are  eight  pairs  of  flagella,  the 
two  posterior  ones  being  continuations  of  longitudinal  axostyles.  The 
nucleus  is  first  dumb-bell  shaped  and  later  divided  into  two  separate 
nuclei.  Cysts  are  found,  and  according  to  Schaudinn  conjugation 
occurs  in  them  with  the  development  of  four  nuclei.  The  young  para- 
sites attach  themselves  to  the  surface  of  epithelial  cells  of  the  small 
intestine  by  the  sucking  disk,  but  even  when  present  in  large  numbers 
do  not  produce  any  characteristic  symptoms.  The  same  or  like  para- 
sites are  present  in  mice,  rats,  dogs,  cats,  and  sheep.  Transmission  is 
by  contact,  as  in  trichomonad  infections. 

ORDER  II— PROTOMONADINA 

The  Protomonadina,  another  order  of  the  Flagellata,  have  less  than 
three  flagella,  and  are  divided  into  the  Cercomonadidce,  Bodonidce  and 
the  Trypanosomidce. 

Genus  1. — Cercomonadidse — Cercomonas  hominis  (Davaine,  1854). 
— As  originally  described,  this  organism  has  a  pear-shaped  body, 
drawn  out  to  a  point  posteriorly,  is  armed  with  a  single  flagellum  in 
front,  and  has  no  undulating  membrane.  It  is  a  doubtful  species  and 
of  no  present  importance. 

Genus  2. — Bodonidae  —  Prowazekia  (Hartmann  and  Chagas). — 
These  organisms,  the  only  examples  of  the  Bodonidce  of  medical  inter- 
est, are  of  some  importance,  since  they  have  been  cultivated  from  hu- 
man feces  on  agar  plates.  The  genus  was  founded  for  Prowazekia 
cruzi,  a  species  discovered  in  human  feces  in  Brazil.  Other  species  are 
urinaria,  asiatica,  parva,  weinhergi  and  javanensis.  There  are  two 
flagella,  arranged  in  the  heteromastigote  manner,  that  is,  one  flagellum 
projects  forward  and  one  trails  behind.  There  is  no  undulating  mem- 
brane, but  in  stained  specimens  a  second  nucleus  is  seen,  the  kineto- 
nucleus  or  blepharoplast.  They  are  also  found  in  water,  and  are 
probably  not  the  cause  of  any  disease. 

Genus  3. — Trypanosomidae. — History  of  the  genus. — In  1841  Val- 
entine discovered  the  first  hemoflagellate  in  the  blood  of  a  trout,  and 
the  following  year  Gruby  described  a  flagellate  in  frog's  blood  and 
named  it  a  ''trypanosome.'^  It  was  not  until  1878  that  Lewis  discov- 
ered the  rat  trypanosome,  Trypanosoma  lewisi.  The  first  pathogenic 
member  of  the  genus  was  noted  by  Evans  in  1889  in  the  blood  of  In- 
dian horses  sick  with  surra,  Trypanosoma  evansi.  Bruce  in  1894  de- 
scribed the  trypanosome  of  Nagana,  a  horse  disease  of  Zululand,  and 


MASTIGOPHORA  741 

also  demonstrated  its  transmission  by  the  tsetse  fly,  Trypanosoma 
hrucei.  In  1894  to  1899,  Rouget,  Schneider  and  Buffard  found  the 
trypanosome  of  Dourine,  or  *'mal  de  coit,"  among  Algerian  horses. 
Elmassian  described,  in  1901,  the  South  American  horse  disease,  ''mal 
de  caderas,''  and  discovered  the  parasite,  Trypanosoma  equinum. 
Since  this  time  a  large  number  of  new  species  have  been  discovered,  the 
more  important  of  which  will  be  described. 

Morphology. — The  morphology  of  the  trypanosomes,  while  subject 
to  many  variations  in  detail,  is  still  uniform  as  to  the  characteristics 
of  the  genus,  so  that  there  is  little  difficulty  in  immediately  recognizing 
the  parasite.  The  body  is  long  and  sinuous,  tapering  anteriorly  to  a 
fine  point  called  the  flagellum ;  the  posterior  end  is  never  so  delicate 
and  is  often  quite  blunt.  All  contain  two  nuclei,  the  larger  being  called 
the  trophonucleus  and  the  smaller  the  kinetonucleus.  The  trophonu- 
cleus  is  usually  located  midway  in  the  length  of  the  body,  and  the 
kinetonucleus  behind  it,  often  at  the  posterior  extremity.  The  flagel- 
lum arises  from  a  centriole  (blepharoplast),  which  is  located  close  to 
or  in  the  kinetonucleus,  and  quickly  reaches  the  surface  of  the  body, 
when  it  turns  forward  and  forms  the  border  of  the  undulating  mem- 
brane, a  thin  fold  of  periblast  running  the  entire  length  of  the  body, 
and  is  often  continued  further  forward  as  delicate  filament.  During 
life  the  undulating  membrane  has  a  constant  wave-like  motion. 

Transmission  from  one  animal  to  another  is  usually  by  means  of 
some  blood-sucking  invertebrate.  Two  possible  forms  of  transmission 
have  been  recognized,  the  direct  and  indirect  or  cyclical;  the  direct 
form  is  used  in  the  laboratory  when  transferring  blood  with  a  hypo- 
dermic syringe  from  an  infected  animal  to  a  healthy  one,  and  it  also 
occurs  in  nature,  although  not  so  frequently  as  the  second.  Dourine, 
or  mal  de  coit,  is  the  best  example  of  the  natural  direct  method.  The 
cyclical  method  is  exemplified  in  the  transmission  of  Trypanosoma 
lewisi  by  the  rat  flea,  Ceratophyllus  fasciatus,  in  which  insect  the  tryp- 
anosome passes  through  a  complicated  life  cycle.  Whether  the  para- 
site in  the  insect  ever  passes  from  parent  to  offspring  is  still  doubtful. 
Among  fishes,  reptiles  and  amphibians  the  parasites  are  carried  by 
leeches,  in  whose  intestinal  tract  they  undergo  a  cycle  of  development. 

Just  as  in  malaria,  there  is  usually  an  alternation  of  hosts,  from 
invertebrate  to  vertebrate,  a  part  of  the  life  cycle  being  passed  in  each. 
In  the  blood  of  the  vertebrate  is  found  the  fully  developed  trypano- 
some, and  in  the  intestinal  tract  of  the  invertebrate,  crithidial  and 
trypanomonad  types,  whicii  are  characterized  by  having  the  kineto- 


742 


PATHOGENIC  PROTOZOA 


nucleus  placed  in  front  of  or  close  beside  the  trophonucleus  and  by 
having  a  rudimentary  undulating  membrane. 

Cultivation. — In  1903,  Novy  and  MacNeal  ^  first  obtained  pure  cul- 
tures of  trypanosomes  on  artificial  media.  The  medium  devisee^  by 
them  is  prepared  by  equal  parts  of  nutrient  agar  and  defibrinated  rab- 
bit blood.  After  the  agar  has  been  melted  and  cooled  to  about  50°  C. 
an  equal  quantity  of  rabbit  blood  is  added,  mixed  and  allowed  to  cool. 
''The  tubes  thus  prepared  are  allowed  to  set  in  an  inclined  position, 
after  which  they  are  at  once  inoculated.  It  is  essential  that  the  sur- 
face of  the  medium  be  moist  and  soft,  and  if  this  is  not  the  case,  the 
tubes  should  be  placed  in  an  upright  position  until  some  water  of 
condensation  accumulates  at  the  bottom.  The  initial  culture  usually 
requires  a  week  or  more,  although  not  infrequently  fairly  rich  growths 
may  be  obtained  in  three  or  four  days"  (Novy). 

Trypanosoma  rotatorium.  —  Gruby  described  and  named  this 
hemoflagellate   in   1843,    and   it   is,   therefore,   the   type   species   of 

the  genus.  The  organism 
is  widely  distributed 
throughout  the  world,  and 
is  found  in  Bana  esculenta, 
Rana  temporaria  and  Hyla 
arhorea;  the  organisms 
are,  however,  not  very  nu- 
merous in  any  single  frog. 
It  is  most  often  found  dur- 
ing the  spring  and  summer 
months,  rarely  in  winter. 
Morphology.  —  Both 
body  and  undulating  mem- 
brane are  broad,  the  cyto- 
plasm is  granular,  and  to- 
w^ard  the  straight  side 
shows  striae,  probably  indicating  the  presence  of  myonemes.  The  tro- 
phonucleus is  large,  lies  near  the  middle  of  the  body  and  near  the 
undulating  membrane;  the  kinetonucleus  is  smaller,  lies  posteriorly 
and  stains  deeply;  the  flagellum  which  originates  near  the  kinetonu- 
cleus turns  forward,  forming  the  border  of  the  undulating  membrane, 


so/t 


Fig.  168. — Trypanosoma  rotatorium  in  Blood 
OF  Frog.  (After MacNeal, ''  Pathogenic  Micro- 
oro;anisms,"  published  by  P.  Blakiston's  Sons 
&  Co.) 


1  Novy  and  MacNeal,  Contrib.  Med.  Research  (Vaughan),  Ann  Arbor,   1903, 
p.  549. 


MASTIGOPHORA 


743 


and  is  continued  forward  as  a  short  flagellum.  The  posterior  end  is 
usually  drawn  out  to  a  stubby  point.  The  fully  developed  organism 
is  large,  being  40  to  80  microns  long  by  5  to  40  wide.  One  striking 
thing  about  this  parasite  is  its  tendency  to  pleomorphism. 

Multiplication  in  the  blood  stream  of  the  frog  is  by  binary  fission ; 
in  addition,  a  form  of  multiple  division  occurs  in  the  viscera,  preceded, 
according  to  Machado,  by  conjugation  of  sexually  differentiated  forms. 
The  merozoites  liberated  from  the  mother  cell  are  small  trypanosomes, 
which  in  turn  grow  to  large  size,  thus  explaining  the  pleomorphism  of 
the  parasite. 

Cultures  have  beon  obtained  by  Lewis  and  Williams  on  the  blood 
agar  of  Novy  and  MacNeal  in  which  a  great  variety  of  forms  may  be 
seen;  the  method  of  transmission  is  unknown,  but  the  infection  is 
probably  conveyed  by  leeches.  Many  other  trypanosomes  have  been 
found  in  fishes,  frogs,  and  reptiles  all  over  the  world. 

Trypanosoina  lewisi  (Kent). — This,  one  of  the  longest  known  and 
commonest  forms,  has  been  studied  more  completely  than  any  other 
organism  of  its  class.  It  occurs  in 
a  large  proportion  of  rats  through- 
out the  world,  twenty-five  to  one 
hundred  per  cent  being  infected, 
and  since  it  is  non-pathogenic,  it  is 
a  convenient  organism  for  research. 
It  may  be  passed  from  wild  to 
white  rats  without  difficulty,  by  in- 
oculating the  latter  with  a  small 
quantity  of  citrated  blood  contain- 
ing the  organisms.  At  first  the 
parasites  are  few,  but  after  the 
lapse  of  three  or  four  days,  large 
numbers  may  be  found ;  the  condi- 
tion of  rapid  multiplication  lasts 
from  eight  to  fourteen  days,  and 
is  succeeded  by  a  period  of  a  month 
or  more,  during  which  time  the 
parasites  gradually  diminish  in 
number,  finally  disappearing  com- 
pletely, rendering  the  animal  immune  from  further  infection,  the 
immunity  being  complete.  The  serum  of  an  immune  rat  has  a  certain 
protective  power,  and  when  inoculated  simultaneously  with  blood  con- 


FiG.  169.  —  Trypanosoma  lewisi. 
(After  Doflein  and  Minchin.  Mac- 
Neal, "Pathogenic  Microorganisms," 
published  by  P.  Blakiston's  Sons  & 
Go.) 


744  PATHOGENIC  PROTOZOA 

taining  trypanosomes,  may  prevent  the  infection.  No  other  animals 
are  susceptible. 

The  blood  should  be  examined  in  both  fresh  and  stained  specimens. 
In  fresh  specimens,  because  of  the  rapid,  lashing  movements  of  the 
parasite,  the  organisms  are  particularly  easy  to  find.  The  details  of 
structure,  however,  do  not  appear  except  in  spreads  stained  with  some 
of  the  modifications  of  the  Romanowski  stain,  such  as  Wright's  or 
MacNeal  's. 

In  the  adult  stage  the  organisms  are  quite  uniform  in  size  and 
shape,  being  27  or  28  microns  long  and  1.5  to  2.0  microns  broad;  the 
posterior  end  is  long,  tapering  and  pointed;  tl^e  kinetonucleus  oval 
and  flattened ;  the  trophonucleus  is  located  near  the  anterior  end,  and 
the  undulating  membrane,  while  distinct,  is  relatively  narrow.  The 
endoplasm  is  finely  granular,  and  by  careful  focusing  the  body  wall  or 
periblast  may  be  seen. 

Multiplication  in  the  rat  is  rapid,  and  many  young  forms  are  seen ; 
these  are  smaller,  stain  more  deeply,  and  vary  much  more  in  size  than 
the  adults.  Dividing  forms  are  common,  the  division  being  longi- 
tudinal and  unequal,  the  parent  retaining  the  flagellum.  Multiple 
division  also  occurs,  resulting  in  the  production  of  rosettes,  whose 
structure  suggests  that  repeated  longitudinal  division  has  occurred 
without  the  separation  of  the  daughter  cells. 

The  insect  hosts  are  two :  the  rat  flea,  Ceratophyllus  fasciatus,  and 
the  rat  louse,  Hcematopinus  spinulosus;  the  former  being  the  right  host 
and  the  latter  the  wrong  one,  since  in  it  development  is  incomplete. 
Minchin  and  Thompson  ^  have  studi-ed  the  cycle  in  the  flea,  which  is 
briefly  as  follows:  When  the  injected  blood  and  parasites  reach  the 
midgut  of  the  flea,  the  trj^panosomes  lose  their  flexibility  and  become 
more  or  less  rigid,  and  are  able  to  penetrate  the  outer  wall  of  the 
epithelial  cells  of  the  stomach.  Once  inside  the  cell,  the  parasite  folds 
upon  itself  and  grows  to  large  size ;  the  nuclei  multiply,  the  body  be- 
comes spherical  and  divides  up  within  its  own  periblast  into  six  or 
eight  daughter  cellsj  all  actively  moving  within  their  common  en- 
velope. This  becomes  tense  and  finally  bursts,  liberating  the  young 
trypanosomes  within  the  epithelial  cell,  through  whose  wall  they  soon 
escape  into  the  lumen  of  the  stomach.  This  form  of  multiplication 
may  be  several  times  repeated,  after  which  the  young  trypanosomes 
pass  down  the  intestine  to  the  lower  end  to  begin  the  rectal  phase. 

1  Minchin  and  Thompson,  Quarter.  Jour.  Micr.  Sc,  Lond.,  1915,  Ix,  463. 


MASTIGOPHORA  745 

There  the  parasites  in  large  numbers  are  found  attached  to  the 
epithelial  cells  by  their  flagella.  Rapid  multiplication  takes  place  by 
repeated  fission  and  the  parasite  becomes  crithidial  in  form,  that  is, 
it  loses  its  undulating  membrane,  becomes  short  and  stubby,  and  the 
kinetonucleus  moveS  forward  close  to  or  in  front  of  the  trophonucleus. 
Ultimately  some  change  back  to  minute  trypanosomes,  and  these, 
when  regurgitated  or  passed  in  the  feces,  serve  to  infect  the  next  vic- 
tim. The  rectal  phase,  when  once  established,  lasts  for  several  months 
or  perhaps  indefinitely,  making  every  infected  flea  a  chronic  carrier. 

Trypanosoma  evansi.  —  Surra  is  a  disease  of  horses  and  mules, 
camels,  elephants,  buffaloes,  and  dogs,  which  prevails  in  India  and 
other  parts  of  Asia,  and  also  in  the  Philippines  and  Northern  Aus- 
tralia. The  Philippine  outbreak  was  traced  to  animals  returned  from 
China  after  the  Boxer  outbreak;  for  at  that  time  American  troops 
came  into  contact  with  native  Indian  troops  and  their  animals. 

The  trypanosome  causing  the  disease  w^as  discovered  by  Evans  in 
1880.  The  clinical  course  of  the  disease  is  marked  by  an  irregular 
recurring  fever,  with  many  remissions,  during  which  the  parasite  can- 
not be  demonstrated  in  the  blood,  although  it  is  not  difficult  to  find 
during  the  febrile  period.  The  animal  is  anemic,  weak,  emaciated, 
and  may  show  an  ecchymotic  eruption  on  the  abdomen.  The  course  of 
the  disease  may  be  either  short  or  long,  but  leads  almost  invariably  to 
death.  In  camels  it  lasts  from  two  to  four  years,  often  without  symp- 
toms until  near  the  end,  and  these  animals  probably  act  as  chronic 
carriers. 

Morphology. — ^Morphologically,  the  parasite  is  very  like  the  Tryp- 
anosoma brucci   of  Nagana,   yet,   as   a  rule,   the  trophonucleus   lies- 
nearer  the  anterior  end  than  in  brucei,  although  it  may  be  impossible 
to  distinguish  in  smears  between  the  two. 

The  disease  is  carried  by  biting  flies,  tahanidce  and' stomoxys,  and 
also  by  fleas. 

Trypanosoma  brucei. — ^Nagana  is  a  well-known  horse  and  animal 
disease  of  Africa,  which  causes  an  enormous  economic  loss  and  has 
greatly  interfered  with  the  development  of  the  country.  The  para- 
site was  discovered  by  Bruce  in  1895.  Among  the  natives  it  is  known 
as  tsetse  fly  disease,  and  investigation  has  incriminated  Glossina  mor- 
sitans  as  the  carrier.  Clinically,  the  disease  in  horses  is  much  like  the 
Surra  of  India;  the  native  name  for  the  disease,  nagana,  means  weak- 
ness. Nearly  all  the  larger  animals  are  susceptible  to  either  natural  or 
artificial  infection,  yet  man  is  apparently  immune.  , 


746 


PATHOGENIC  PROTOZOA 


Morphology. — Morphologically,  it  resembles  closely  most  of  the 
other  pathogenic  trypanosomes,  and  Minchin  makes  it  the.  type  of  a 
group  of  pathogenic  trypanosomes,  all  closely  resembling  one  another 
and  possibly  descended  from  one  common  ancestor :  the  group  consists 
of  hrucei,  gambiense,  evansi,  equiperdum,  rhodesUnse,  and  hippicum. 
The  organism  is  less  slender  than  lewisi  and  has  a  wider  undulating 
membrane.  The  posterior  end  is  relatively  short,  the  trophonucleus 
lies  in  the  middle  of  the  body  and  the  kinetonucleus  at  the  extreme  pos- 


B 


«^ 


Fig.  170. — The  Most  Important  Trypanosomes  Parasitic  in  Vertebrates.  A, 
Tr.  lewisi;  B,  Tr.  evansi  (India);  C,  Tr.  evansi  (Mauritius);  D,  Tr.  brucei;  E, Tr. 
equiperdum;  F,  Tr.  equinum;  G,  Tr.  dimorphon;  H,  Tr.  gambiense.  (X  1500.) 
(From  Doflein  after  Novy.  MacNeal,  ''Pathogenic  Microorganisms,"  published 
by  P.  Blakiston's  Sons  &  Co.) 

terior  end;  a  vacuole  is  placed  just  in  front  of  the  latter.  In  length 
the  parasite  measures  twenty-five  to  thirty-five  microns  and  is  one  and 
a  half  to  two  and  a  half  microns  in  width ;  multiplication  in  the  blood 
stream  is  by  binarj'-  fission. 

Transmission  is  by  means  of  the  tsetse  fly,  Glossina  morsitans,  and 
perhaps  Glossina  pallidipes  and  Glossina  fusca.  The  fly  may  transmit 
the  disease  directly  after  infection,  acting  as  a  mere  mechanical  car- 
rier, but  it  is  more  probable  that  a  cyclical  development  of  the  para- 
site takes  place  in  the  fly,  after  which  it  remains  infectious  for  a  long 


MAStlGOPHORA  U7 

period.  It  has  been  shown  that  after  the  first  few  hours  the  fly  is  not 
infectious  again  until  the  lapse  of  eighteen  days,  when  its  bite  once 
more  conveys  the  disease,  and  trypanosomes  may  be  found  in  the  intes- 
tinal canal,  the  body  cavity,  the  salivary  glands  and  in  the  proboscis. 
Studies  of  the  cycle  in  the  fly  show  that  only  about  five  per  cent  of 
the  flies  permitted  to  feed  on  sick  animals  become  chronic  carriers. 
The  work  of  Bruce  and  others  has  shown  that  the  trypanosomes  are 
more  or  less  harmless  parasites  of  the  big  game  animals  of  Africa, 
w^hich  therefore  are  believed  to  act  as  a  reservoir,  from  which  the  dis- 
ease is  transferred  to  the  domestic  animals  by  the  tsetse  fly.  The  dis- 
tribution of  Glossina  is  not  uniform,  as  they  are  only  present  in  cer- 
tain definite  areas  called  fly  belts.  Since  the  disease  does  not  spread 
in  the  absence  of  the  larger  wild  animals,  it  has  been  proposed  that  all 
big  game  be  exterminated  as  a  prophylactic  measure.  Mice  and  rats 
are  susceptible  and  die  in  six  to  fourteen  days  after  inoculation; 
guinea-pigs  are  more  resistant,  and  may  show  one  or  more  relapses 
within  two  to  ten  weeks.  It  has  not  been  possible  to  immunize  larger 
animals,  although  a  certain  degree  of  success  has  been  obtained  with 
the  smaller  animals  used  in  the  laboratory. 

Cultures  have  been  grown  on  artificial  media,  yet  not  so  readily 
as  with  lewisi  and  avian  trypanosomes.  The  medium  recommended  by 
MacNeal  contains  the  extractives  of  one  hundred  and  twenty-five  grams 
of  meat,  ten  of  peptone,  five  of  salt,  and  twenty -five  of  agar  to  the 
liter;  to  this  is  added  twice  its  volume  of  warm,  defibrinated  rabbit's 
blood.  The  blood  agar  slants  should  be  soft  and  moist  when  inocu- 
lated. Filtrates  from  cultures  are  not  toxic,  the  toxin  apparently 
being  liberated,  according  to  MacNeal,  from  the  body  of  the  disinte- 
grating trypanosome. 

Trypanosoma  hippicum  (Darling). — The  disease  caused  by  this 
trypanosome  in  horses  and  mules  has  been  known  in  Panama  for  many 
years  under  the  name  of  '^Murrina  de  caderas"  or  "  Derrengadera  de 
caderas,''  the  latter  term  being  used  when  paralysis  of  the  posterior 
extremities  is  the  dominant  symptom ;  both  names  indicate  a  weakness 
of  the  hind  quarters.  The  symptoms  are  weakness,  emaciation,  and, 
sooner  or  later,  conjunctivitis  and  subconjunctival  ecchymosis,  and 
anemia.  The  horses  and  mules  affected  are  obviously  weak,  and  while 
in  the  stall,  pull  back  on  the  halter,  or  stand  with  straddling  hind  legs. 

The  incubation  period  in  animals  used  for  experiment  is  less  than 
a  week,  a  few  animals  lose  weight  rapidly  and  die  within  a  few  days, 
others  live  for  several  weeks. 


748  PATHOGENIC  PROTOZOA 

Treatment,  including  the  use  of  arsenical  preparations,  is  without 
effect,  and  all  infected  animals  should  be  destroyed. 

The  disease  is  apparently  transmitted  directly  by  flies,  which  carry 
blood  and  serum  from  ulcers  and  abrasions  on  infected  to  healthy 
animals. 

Morphology  of  the  Parasite. — The  trypanosome  is  sixteen  to  eight- 
een microns  long  and  two  microns  wide.  The  kinetonucleus  is  about 
two  microns  from  the  posterior  end,  the  trophonucleus  about  eight  to 
ten  microns  from  the  same  point.  The  posterior  end  is  blunt  and  the 
cytoplasm  usually  contains  numerous  basophile  granules;  the  undu- 
lating membrane  is  well  developed  and  a  chromatin  filament  runs  from 
the  kinetonucleus  to  the  tip  of  the  flagellum.  The  large  kinetonucleus 
distinguishes  this  organism  from  Trypanosoma  equinum. 

Pathological  Anatomy. — Aside  from  the  emaciation,  edema  of  the 
belly  wall,  conjunctivitis  and  subconjunctival  ecchymosis,  there  is  usu- 
ally excessive  fluid  in  the  body  cavities,  an  enlarged  spleen,  and,  what 
is  more  characteristic,  small  petechial  spots  on  the  capsule  of  the 
spleen  and  in  the  cortex  of  the  kidney  and  in  the  endo-  and  pericar- 
dium and  occasionally  on  the  pleural  surfaces. 

Prophylaxis  consists  in  the  destruction  of  all  infected  animals ;  the 
protection  of  wounds  and  ulcers  in  otherwise  healthy  animals  by  dress- 
ings and,  wherever  possible,  the  use  of  fly  screens  about  the  stables. 

Trypanosoma  eqiiiperdum  (Dourine). — This  organism  is  the  cause 
of  dourine,  a  disease  of  horses  and  donkeys,  which  is  usually  trans- 
mitted by  coitus,  but  may  be  carried  by  biting  fliies,  stomoxys.  The 
organism  was  first  described  by  Rouget  in  1894 ;  it  resembles  hrucei  in 
many  ways  and  produces  a  progressive,  fatal  disease  of  great  economic 
importance.  Formerly  it  was  present  throughout  the  greater  part  of 
Europe,  but  is  now  almost  limited  to  the  shores  of  the  Mediterranean. 
From  time  to  time  it  has  been  introduced  into  the  United  States  and 
Canada  by  blooded  French  stallions  and  has  spread  into  parts  of  the 
Northwest. 

The  clinical  course  may  be  divided  into  a  stage  of  edema,  lasting 
about  a  month,  during  which  there  is  a  painless,  soft  swelling,  limited 
to  the  genitalia  and  the  belly  wall.  This  is  followed  by  the  stage  of 
eruption,  during  which  plaques,  or  round  edematous  areas,  are  found 
under  the  hide  on  the  flanks  and  hind  quarters,  and  sometimes  on 
thighs,  shoulders  and  neck;  this  stage  is  short,  lasting  about  a  week. 
It  is  followed  by  the  third  stage  of  paralysis  and  anemia ;  the  animal 
loses  flesh   and  strength,   develops  superficial  ulcers,  conjunctivitis, 


MASTIGOPHORA  749 

keratitis,  and  ultimately  paralysis,  leading  to  death  in  two  to  eighteen 
months. 

The  trypanosome  is  found  most  readily  in  the  serous  exudate  from 
the  ulcers,  as  it  is  infrequent  in  the  peripheral  circulation;  in  this 
respect  it  resembles  the  treponema  of  lues.  The  organism  is  about 
twenty-five  microns  in  length  and  possesses  a  clear  cytoplasm,  free 
from  granules,  except  when  propagated  in  white  mice,  when  they  are 
plentiful. 


Fig.  171. — Dourine.  Showing  swelling  of  genitalia  and  plaques  on  the  skin. 
(After  KoUe  and  Wassermann,  "Handbuch  der  Pathogenen  Mikro-organismen," 
2te  Aufl.,  1913.) 

Diagnosis  hy  Complement  Fixation. — E.  A.  Watson,^  of  Canada, 
has  shown  that  it  is  possible  not  only  to  diagnose  the  disease  when  the 
clinical  signs  are  clear,  but  also  to  determine  the  existence  of  its  non- 
clinical, obscure  and  latent  forms.  Horses  may  tolerate  an  infection 
for  one  to  three  years,  during  which  time  they  are  capable  of  convey- 
ing the  disease  and  yet  remain  normal  in  health  and  general  appear- 
ance, and  this  method  of  diagnosis  is,  therefore,  invaluable. 

Watson  obtains  the  antigen  by  inoculating  a  large  number  of 
white  rats  with  Trypanosoma  equiperdum,  collecting  their  blood  when 

^E.  A.  Watson,  Parasitology^  Cambridgs,  Enp.,  1915,  VIII,  156. 


750 


PATHOGENIC  PROTOZOA 


teeming  with  trypanosomes,  and  separating  them  from  the  erythro- 
cytes and  plasma  by  washing  and  centrifuging.  Each  of  ten  to  twenty 
rats  receive  0.3  c.c.  of  blood  rich  in  trypanosomes  intraperitoneally, 
and  at  about  the  end  of  the  third  day,  when  the  organisms  are  very 
numerous,  the  rats  are  bled  into  citrate  solution.  By  repeated  washing 
the  organisms  may  be  separated,  as  a  pure  white  layer  overlying  the 
erythrocytes.  This  mass  of  organisms  is  killed  and  preserved  by  a 
formalin-glycerin  mixture,  after  which  its  antigenic  strength  is  stand- 
ardized by  titration  in  the  usual  Avay.  The  test,  a  pure  culture  of 
trypanosomes  being  used  as  antigen,  is  specific  and  is  not  positive 
in  any  other  disease  of  horses. 

Trypanosoma  avium. — This  parasite  was  first  described  by  Dani- 
lewski  in  1885.     In  1905,  Novy  and  MacNeal  ^  found  trypanosomes 


Fig.  172. — Trypanosoma  avium  in  Blood  of  Common  Wild  Birds.  (After  Novy 
and  MacNeal.  MacNeal,  "Pathogenic  Microorganisms,"  published  by  P.  Blakis- 
ton's  Sons  &  Co.) 


in  8.8  per  cent  of  431  American  birds.  Although  there  are  doubt- 
less several  species,  the  most  common  is  Trypanosoma  avium,  a  para- 
site twenty  to  seventy  microns  long  and  four  to  seven  microns  wide. 
They  are  found  in  the  blood  over  long  periods  of  time  and  do  not 
appear  to  be  pathogenic.  Cultures  are  easily  made  and  kept  alive 
for  long  periods  by  weekly  transfers.  The  mode  of  transmission  is 
unknown. 

This  was  the  parasite  which  was  confounded  in  1904  by  Schaudinn 
with  developmental  stages  in  the  life  cycle  of  Hemorproteus  noctiice 

1  Novy  and  MacNeal,  Jour.  Infect.  Dis.,  Chicago,  190.5,  ii,  256. 


MASTIGOPHORA 


751 


and  Hemorproteus  ziemani  with  resulting  confusion  in  the  study  of 
trypanosomes  and  hemoeytozoa,  and  it  is  only  recently  that  the  error 
has  been  generally  ackowledged. 


Fig.  173. — Trypanosoma  avium  in  Culture  on  Blood  Agar.  (X  1500.)  (After 
Novy  and  MacNeal.  MacNeal,  "Pathogenic  Microorganisms,"  published  by  P. 
Blakiston's  Sons  &  Co.) 

Trypanosoma  gambiense  (Sleeping  Sickness). — Two  names  have 
been  given  to  the  disease  caused  by  this  parasite,  both  of  which  are 
now  recognized  as  stages  in  one  and  the  same  infection,  human  tryp- 
anosomiasis: they  were  trypanosome  fever,  and  sleeping  sickness.  It 
is  a  chronic  infection  characterized  by  fever,  lassitude,  weakness, 
wasting,  and,  in  its  terminal  stages,  by  a  protracted  lethargy.  Sleep- 
ing sickness  and  trypanosome  fever  had  long  been  known  in  tropical 
Africa,  and  the  disease  at  present  is  widespread  and  the  cause  of 
tremendous  mortality.  It  is  estimated  that  one  hundred  thousand 
deaths  accurred  during  the  ten  years  ending  in  1910.     It  is  endemic 


752 


PATHOGENIC  PROTOZOA 


in  the  lake  region  of  Central  Africa,  and  in  the  Congo  basin.  It 
was  early  introduced  into  Martinique  in  the  West  Indies,  but  did  not 
spread  and  has  now  died  out. 

Dutton  and  Todd  found  the  parasite  in  1901  in  the  blood  of  an 
Englishman  in  Gambia,  who  died  after  a  febrile  illness  of  two  years' 


Fig.  174. — Trypanosoma  gambiense.    Calkin,  "Protozoology." 


duration ;  Castellani  in  1903  found  the  parasite  in  the  cerebro-spinal 
fluid  of  well-marked  cases  of  sleeping  sickness  occurring  among 
natives  of  Uganda. 

Clinical  Signs. — The  disease  begins  with  slight  febrile  attacks, 
headache  and  increasing  weakness,  emaciation,  swelling  of  the  eyelids 
and  enlargement  of  the  lymph  nodes.  The  temperature  increases, 
edema  of  the  extremities  appears  and  the  spleen  enlarges.  During 
the  last  stages  nervous  symptoms  predominate  and  the  patient  sleeps 
day  and  night,  but  may  have  periods  of  excitement  or  convulsions, 
y.et  finally  sinks  into  deep  coma  and  dies  of  exhaustion. 

Etiology. — The  disease  is  transmitted  by  the  bite  of  the  tsetse  fly, 
Glossina  palpalis,  which  is  apparently  able  to  transmit  the  infection 
mechanically  immediately  after  biting  an  infected  host,  yet  in  most 
flies  the  trypanosomes  disintegrate  and  disappear  from  the  intestinal 
tract  within  four  or  five  days.   Jn  from  Jive  .to -ten  per  cent  of  the 


Mastigophora 


■753 


Fig.  175. — Tsetse  Fly  (Glossind  pnlpalis).     (From 
Rosenau)  "Preventive  Medicine  and  Hygiene.") 


flies,  however,  the  trypanosomes  multiply  in  the  intestinal  tract,  and 
after  eighteen  to  fifty -three  days  they  again  become  infectious  and  re^ 
main  so  for  a  long 
period,  the  parasites  be- 
ing found  regularly  in 
the  salivary  glands  and 
in  the  proboscis. 

It  is  possible  that  the 
disease  is  transmitted  in. 
other  ways  than  by 
Glossina  palpalis ;  blood- 
.sucking  insects,  such 
as  stomoxys,  anopheles, 
mansonia  and  perhaps 
fleas,  may  act  as  me- 
chanical carriers.  It  is 
also  possible  that  the 
disease  is  transmitted 
by  coitus.  Without  some 

isuch  explanation  it  is  difficult  to  understand  certain  house  epidemics 
which  have  occurred  outside  the  fly  belts. 

The  animal  host  of  the  Trypanosoma  gamhiense  is  believed  to  be 
the  big  game  animals,  particularly  the  antelope. 

Morphology. — The  organism  belongs  to  the  brucei  group,  and  its 
'differentiation  on  morphology  is  difficult,  yet,  on  the  average,  the 
posterior  end  is  somewhat  more  pointed  than  the  hrucei.  In  length 
it  varies  from  fifteen  to  thirty  microns,  and  in  thickness  from  one  to 
three  microns.  In  fresh  preparations  the  motility  is  not. marked; 
both  plump  and  slender  forms  are  found  in  the  blood,  but  in  the  eere- 
bro-spinal  fluid  slender  forms  only  are  seen. 

Cultures  on  blood  agar  have  been  made  by  Thompson  and  Sinton, 
yet  they  died  out  after  a  few  weeks,  and  were  never  virulent.  The 
pathogenicity  varies  somewhat  with  the  strain  used,  but  apes  are  easily 
infected.  In  white  rats  there  may  be  two  or  three  relapses  before 
death  occurs,  while  when  inoculated  with  hrucei  death  follows  within 
two  weeks. 

Pathogenicity. — Although  cultures  vary  greatly  in  virulence,  it  is 
possible  to  infect  rats,  dogs  and  monkeys  with  a  fatal  trypanosomiasis ; 
cattle,  sheep  and  goats  continue  to  show  a  few  parasites  for  months 
after  inoculation  but  without  sickening.     In  no  animal,  however,  is 


754  PATHOGENIC  PROTOZOA 

it  possible  to  reproduce  the  sleeping  sickness  stage  as  it  occurs  in 
man. 

Diagnosis. — When  the  disease  is  well  developed  in  an  endemic 
area,  the  diagnosis  is  easily  made.  During  the  early  stages  the  exami- 
nation of  the  cerebro-spinal  fluid,  puncture  fluid  from  the  lymph  nodes 
and  the  peripheral  blood  may  all  show  the  trypanosome;  since  the 
parasites  are  scarce  the  use  of  the  thick  film  method  of  Ross  may  be 
necessary.  When  direct  examination  is  unsuccessful,  enrichment  in 
the  blood  of  susceptible  animals,  rats  and  mice  will  establish  the 
diagnosis. 

Treatment. — Treatment  is  based  upon  the  observation  of  Bruee 
and  Lingard,  that  arsenious  acid  is  trypanocidal.  The  best  results 
have  been  obtained  with  atoxyl,  in  half  gram  doses,  repeated  at  inter- 
vals of  ten  days  or  more  for  not  less  than  four  months.  Light  cases: 
become  trypanosome  free  and  are  apparently  cured,  yet  many  relapse 
on  cessation  of  treatment.  Well  marked  cases  may  show  improvement; 
yet  ultimately  grow  worse  and  die.  Other  arsenical  preparations  have 
been  used  but  none  are  entirely  successful.  Salvarsan  drives  the 
parasite  from  the  peripheral  blood  but  not  from  the  cerebro-spinal 
fluid.    The  prognosis  is  unfavorable. 

Prophylaxis. — Prophylaxis  is  quite  complicated  and  is  carried  out 
along  several  different  lines.  Infected  fly  belts  are  depopulated,  the 
inhabitants  being  removed  to  a  fly-free  district  where  they  may  be 
treated  at  hospital  stations.  The  fly  breeding  may  be  greatly 
diminished  by  clearing  off  the  forest  and  brush,  especially  along  the 
river  courses,  since  the  glossina  needs  abundant  moisture  for  its  propa- 
gation. Since  the  fly  bites  only  during  the  day,  all  traveling  in 
infected  districts  is  best  done  at  night. 

As  it  is  recognized  that  the  antelope  is  the  permanent  reservoir  for 
Trypanosoma  gambiense,  the  obvious  remedy  is  its  extermination. 

Trypanosoma  rhodesiense.  —  This  parasite  was  established  hy 
Stephens  and  Fantham.^  It  is  transmitted  by  the  Glossina  morsitans, 
a  fly  which  is  widespread  over  large  tracts  of  country,  independently 
of  the  presence  of  water.  It  is  becoming  generally  recognized  that 
there  are  two  forms  of  sleeping  sickness,  one  of  which  is  caused  by 
this  trypanosome.  This  form  of  the  disease  is  more  acute  and  is 
unaffected  by  treatment;  the  trypanosome  is  also  more  virulent  for 
animals  and  may  be  differentiated  from  gambiense  on  its  morphology. 

*  Stephens  and  Fantham,  Proc.  Roy.  Soc,  1910,  Ser.  B.,  Ixxxiii,  28. 


MASTIGOPHORA  755 

As  both  parasites  are  found  in  the  antelope,  the  prophylaxis  is  the 
same.  Bruce  ^  is  of  the  opinion  that  rhodesiense  and  brucei  are 
identical. 

Schizotrypanum  cruzi.  —  This  parasite,  which  differs  from  all 
other  trypanosomes,  is  the  cause  of  a  form  of  human  trypanosomyasis 
occurring  in  Brazil.  It  is  transmitted  by  a  bug,  Conorhinus  megistus, 
in  which  the  parasite  passes  part  of  its  cycle  of  development.  In  the 
human  being,  multiplication  takes  place  in  endothelial  cells,  lympho- 
cytes and  other  parenchymatous  cells  of  the  viscera;  and  also  in  the 
skeletal  and  heart  muscles.  While  in 
this  stage  the  parasite  has  no  flagel- 
lum  and  resembles  the  leishmania; 
only  after  escape  into  the  blood  does 
it  take  on  the  trypanosome  form. 

Guinea-pigs,  rats,  mice  and  mon- 
keys are  susceptible ;  the  bed-bug,  Fig.  176.— Schizotrypanum  cruzi 
cimex,  is  also  capable  of  transmitting  in  Human  Blood.  (From  Dof- 
the   disease  ^^^^  after  Chagas.  MacNeal,  Path- 

n    1.  1^2.   '      ;j  T^     nv.  ogenic     Microorganisms,"     pub- 

Cultures  were  obtained  by  Chagas         j.^^^  j^^  p.  Blakiston's  So^  & 
and  proved  virulent  for  animals.  The         Qq  ) 
human  disease  is  found  both  in  chil- 
dren and  adults  and  is  regularly  fatal.     It  is  characterized  by  an 
irregular  fever,  severe  anemia,  swelling  of  the  lymph  nodes,  edema 
and  disturbance  of  the  nervous  system. 

Leishmania. — This  genus  was  founded  by  Ross  in  1903  for  the 
Leish man-Donovan  and  Wright  bodies  found  in  kala-azar  and  Delhi 
boil,  to  which  Nicolle  added  another  in  1909,  the  parasite  of  infantile 
splenomegaly.  Leishman,  Donovan  and  Wright,  working  independ- 
ently, described  the  first  two  parasites  in  1903,  and,  although  they  have 
received  various  names,  leishmania  is  now  the  accepted  term.  Rogers, 
Calkins  and  others,  however,  class  them  a^  herpetomonads,  because  of 
the  elongated,  flagellated  form  all  take  in  cultures  on  the  Novy-Mac- 
Neal-Nicolle  blood  agar  medium.  It  is,  however,  best  to  consider  them 
as  a  separate  genus,  because  of  their  natural  parasitic  habits  in 
human  beings.  Leveran,  Fantham  and  others  have  shown  that  it  is 
possible  in  the  laboratory  to  induce  the  herpetomonads  parasitic  in 
the  intestine  of  various  insects  to  become  parasitic  in  various  ver- 
tebrates. 

1  Bruce,  Bull.  Trop.  Dis.,  1916,  vii,  68. 


756 


PATHOGENIC  PROTOZOA 


^W^~ 


\ 


2 


RLShcppako. 


Fig.  177. — Schizotrypanum  cruzi  Developing  in  Tissues  of  Guinea-pig.  1, 
Cross-section  of  fibers  of  striated  muscle  containing  Schizotrypanum  cruzi;  2, 
Section  of  brain  showing  cyst  in  a  neurogUa  cell  containing  chiefly  flagellated 
forms;  3,  Section  through  suprarenal,  fascicular  zone;  4,  Section  of  brain  show- 
ing neuroglia  cell  filled  with  round  forms,  (After  Low  and  Vianna.  MacNeal, 
"  Pathogenic  Microorganisms,  published  by  Blakiston's  Sons  &  Co.) 


MASTIGOPHORA 


757 


Leishmania  donovani  (Kala-azar).— This  parasite  is  the  cause  of 
kala-azar,  a  disease  characterized  by  irregular  fever,  weakness,  anemia, 
cachexia  and  a  remarkable  enlargement  of  the  spleen,  and  occasionally 
of   the   liver.      It   is 
chronic,     progressive 
and  frequently  fatal, 
the    mortality    being 
about    80    per    cent. 
The   disease  .is   com- 
mon in  tropical  Asia 
and   in   northeastern 
Africa. 

Morphology. — The 
parasite  is  intracellu- 
lar, and  is  found 
principally  in  the 
endothelial  cells  of 
the  spleen  and  liver, 
and  in  the  bone  mar- 
row. It  is  oval,  two 
to  four  microns  in  di- 
ameter, finely  granu- 
lar and  occasionally 
vacuolated.  It  con- 
tains a  large,  round 
nucleus  and  a  smaller 
blepharoplast  which 
is  oval  or  rod  shaped ; 
a  third  body,  a  slen- 
der short  thread,  may  sometimes  be  recognized,  which  is  presum- 
ably the  undeveloped  flagellum.  Stained  specimens  of  blood,  spleen 
and  liver  pulp,  and  bone  marrow,  usually  show  large  endothelial  celk 
or  leucocytes  closely .  packed  with  parasites,  one  to  two  hundred  to  a 
single  cell.  Multiplication  in  the  body  is  by  simple  division,  and 
incompletely  divided  pairs  of  organisms  are  frequently  seen.  Cultures 
have  been  obtained  in  citrated  blood  and  on  the  usual  N.  N.  N.  medium. 
When  fully  grown  the  cultural  organisms  are  typical  herpetomonads 
(leptomonads)  ;  the  cell  body  elongates  and  the  rudimentary,  whip 
develops  into  a  true  flagellum.  Both  dogs  and  monkeys  are  susceptible 
to  artificial  inoculations. 


Fig. 


178. — Leishmania    donovani.       (Army 
School  Collection,  Washington,  D.  C.) 


Med. 


758  PATHOGENIC  PROTOZOA 

The  parasite  is  probably  transmitted  by  some  insect,  either  cimex 
(Rogers),  or  by  the  dog  flea,  Ctenocephalus  canis  (Wenyon),  or  a 
plant-feeding  bug,  Conorhinus,  which  occasionally  sucks  blood. 

Animal  Pathogenicity. — Wenyon  in  1913  ^  inoculated  a  dog  with 
splenic  emulsion  from  a  man  who  died  in  London  of  kala-azar  con- 
tracted in  Calcutta.  The  parasite  has  been  successfully  carried 
through  five  animals,  and  in  1915  an  examination  of  the  bone  marrow 
showed  not  only  typical  leishmania,  but  also  a  few  large,  well-marked 
leptomonad  forms.  Similar  forms  were  described  by  Escomel  in  1911, 
from  South  American  dermal  lesions.    Monkeys  may  also  be  infected. 

Leishmania  tropica  (Delhi  or  Aleppo  boil)  is  the  organism  found 
in  a  local  skin  affection  variously  termed  Delhi  boil,  Aleppo  boil  or 
tropical  ulcer.  While  it  is  probably  transmitted  by  some  insect,  there 
is  as  yet  no  definite  proof.  The  incubation  period  is  about  two 
months,  while  the  disease,  once  manifest,  lasts  twelve  to  eighteen 
months  and  is  followed  by  immunity  for  life. 

The  parasite,  which  was  first  described  by  J.  H.  Wright,^  shows 
minor  differences  from  Leishmania  donovani,  particularly  a  variable 
morphology,  all  gradations,  from  the  usual  oval  to  elongated  narrow 
forms  with  pointed  ends,  being  found. 

Cultures  may  be  obtained  on  the  N.  N.  N.  blood  agar,  which  develop 
into  leptomonads,  as  with  Leishmania  donovani.  Dogs  and  monkeys 
are  susceptible  to  artificial  inoculation,  and  it  is  possible  that  in  nature 
the  disease  is  carried  from  dogs  to  human  beings  by  some  insect. 

Leishmania  infantum  (Infantile  Splenomegaly)  was  described 
by  Nicolle  in  1909  from  cases  of  infantile  splenomegaly  occurring  in 
Northern  Africa.  The  disease  resembles  kala-azar  in  all  respects,  except 
that  the  patients  are  young  children,  and  it  is  possible  they  are  the 
same  disease.  The  parasites  are  found  in  abundance  in  the  liver, 
.spleen  and  bone  marrow  at  autopsy  and  may  be  cultivated  in  the  usual 
way  on  the  N.  N.  N.  blood  agar. 

Animal  Pathogenicity. — The  disease  occurs  naturally  in  African 
dogs,  and  they  are  probably  the  source  of  infection,  the  parasite  being 
carried  by  a  flea  or  some  other  insect.  Dogs,  monkeys  and  guinea-pigs 
are  susceptible  to  artificial  inoculation. 

The  treatment  of  leishmaniasis  is  unsatisfactory  since  there  is  no 
known  specific. 


1  Wenyon,  Jour.  Trop.  Med.  and  Hyg.,  London,  1915,  xviii,  218. 

2  Wnght,  J.  H.,  Jour.  Med.  Res.  Bost.  1903,  x,  472. 


MASTIGOPHORA  759 

Two  other  forms  of  dermal  leishmaniasis  have  been  described ;  the 
first,  due  to  Leishmania  hraziliensis,  occurs  in  many  parts  of  South 
America.  The  parasite  is  morphologically  identical  with  Leishmania 
tropica.  Since  the  disease  is  always  contracted  in  the  virgin  forest, 
one  name  for  the  affection  is  forest  yaws ;  uta  and  espundia  are  prob- 


V 

f 

m 

Fig.  179. — Leishmania  infantum.     (Army  Med.  School  Collection,  Washington, 

D.  C.) 

ably  different  clinical  forms  of  the  same  disease.  The  transmitting 
insect  cannot  well  belong  to  the  household  vermin  or  domestic  insects ; 
sylvan  insects  such  as  the  ixodides,  tabanides,  simulids,  mosquitoes  and 
Conorhinus  are  all  suspected  of  being  carriers. 

The  second  form  is  called  Leishmania  nilotica  (Brumpt,  1913), 
and  is  found  in  non-ulcerating  keloid  ijodules  in  Egyptian  negroes. 
Morphologically,  the  parasite  is  indistinguishable  from  Leishmania 
tropica. 


CHAPTER   LVII 

CLASS  III— SPOROZOAi 
SUB-CLASS— TELOSPORIDIA 

HEMOSPORIDIA 

The  Hemosporidia  and  Sarcosporodia  are  the  only  members  of  this 
order  of  medical  interest.  The  hemosporidia  belong  to  the  sub-class 
Telosporidia  of  the  Sporozoa,  because  spore  formation  begins  at  the 
end  of  the  life  cycle. 

The  systematists  have  not  yet  agreed  upon  the  proper  classification 
of  this  group  of  parasites;  consequently  the  older  arrangement  will 
be  followed.  They  are,  like  the  coccidia,  parasites  of  cells,  at  least 
during  the  schizogenous  cycle ;  all  change  hosts  to  some  insect  for  the 
sporogenous  cycle.  As  the  name  implies,  they  live  in  blood  cells  and 
are  rapidly  growing  ameboid  bodies,  which,  beginning  as  sporozoites, 
penetrate  the  host  cells  and  develop  into  trophozoites.  These  grow 
rapidly  to  adult  segmenting  parasites,  in  which  case  they  are  called 
schizonts,  or  to  sexual  forms,  or  gametes,  when  they  are  termed 
sporonts.  In  the  course  of  their  development,  most  species  produce 
melanin  from  the  destruction  of  the  hemaglobin. 

The  nucleus,  which  is  readily  stained,  is  single  and  possesses  a 
karyosome ;  the  mature  schizont  divides  into  many  small  forms  called 
merozoites,  and  these,  when  freed  by  the  rupture  of  the  degenerated 
erythrocyte,  escape  into  the  blood  plasma,  and  if  not  phagocyted, 
penetrate  other  erythrocytes  and  repeat  the  asexual  or  schizogenous 
cycle.  The  pigment  and  undivided  portion  (restkorper)  of  the  cyto- 
plasm of  the  mother  cell  accumulate  in  the  bone  marrow,  spleen  and 
other  viscera. 

After  a  number  of  cycles  of  asexual  multiplication  have  been  lived 
through,  a  new  development  takes  place  and  sexual  forms  begin  to 
appear  in  the  circulation.  These  grow  to  large  size,  yet  show  no  indi- 
cation of  division  into  merozoites  and  were  at  one  time  considered 
degeneration  forms.     Two  varieties  may  be  distinguished,  one  with  a 

1  For  classification,  see  page  722, 
760 


SPOROZOA  761 

dark  staining  cytoplasm  and  fine  granular  melanin,  and  the  other  with 
light  staining,  hyaline  cytoplasm  and  coarse  pigment;  the  former, 
loaded  with  reserve  food  material,  is  the  female  or  macrogametocyte ; 
the  latter,  the  male  or  microgametocyte.  The  gametes  do  not  develop 
further  until  taken  into  the  digestive  tract  of  the  insect  host.  For 
purposes  of  study,  however,  the  microgametocytes  may  be  made  to  ex- 
flagellate  on  the  slide,  dampened  a  little  by  breathing  upon  it,  to 
stimulate  the  condition  in  the  insect  host.  In  such  a  preparation,  the 
flagella,  or  microgametes,  may  be  seen  actively  moving  inside  the  cell 
body,  whose  wall  they  ultimately  rupture,  and  all,  four  to  eight,  escape 
and  whip  about  until  they  come  in  contact  with  a  macrogametocyte, 
when  one  microgamete  enters  through  the  micropyle  and  finally  fuses 
with  the  female  nucleus. 

Hemoproteus  columbse  (Halteridium). — This  parasite  of  the  red 
blood  cells  of  doves  was  described  in  1891  by  Celli  and  Sanfelice. 
It  is  widely  distributed  in  nature  and  has  been  reported  from  Europe, 
Asia  and  North  and  South  America.  The  organism  is  found  within 
the  cyptoplasm  of  the  erythrocyte ;  the  nucleus,  which  is  not  regularly 
displaced,  is  surrounded  by  the  growing  parasite  like  a  halter,  and 
for  this  reason  it  was  named  halteridium  by  Labbe.  It  is  sluggishly 
ameboid  and  produces  an  abundance  of  melanin,  and  when  the  blood 
is  drawn  the  ripe  male  sporonts,  the  microgametocytes.  rupture  easily, 
liberating  the  active  flagella,  or  microgametes.  Under  favorable  cir- 
cumstances the  fertilization  of ,  the  macrogametocyte  by  the  micro- 
gametes may  be  observed  on  the  slide,  and  it  was  while  working  with 
this  parasite  that  Macallum  first  followed  out  the  whole  process  of 
fertilization  in  the  hemosporidia  and  gave  the  proper  explanation  of 
the  flagellate  stage  seen  in  the  malarial  parasite. 

In  the  blood  of  the  dove  this  parasite  is  usually  seen  as  a  large  or 
small  crescent,  partly  encircling  the  nucleus ;  the  gametes  are  readily 
recognized  by  the  usual  marks,  that  is,  the  female,  or  macrogametocyte, 
is  rich  in  reserve  material  and  the  stained  specimen  takes  a  deep  color ; 
the  male,  or  microgametocyte,  being  poor  in  reserve  material  stored 
in  the  cyptoplasm,  appears  relatively  pale  in  stained  specimens. 

The  invertebrate  host  of  the  parasite  is  Lynchia  maura  (Bigot), 
or  Lynchia  lividocolor,  a  biting  hippoboscid  fly  of  louse-like  habits 
which  lives  in  the  nest  and  in  the  plumage.  The  cycle  in  the  fly  has 
been  successfully  worked  out  by  Adie,^  who  has  demonstrated  the 

*  Adie,  Helen,  Indian  Jour.  Med.  Research,  Calcutta,  1915. 


762 


PATHOGENIC  PROTOZOA 


ookinetes,  zygotes  and  oocysts  in  the  lower  portion  of  the  midgut.  As 
the  oocyst  grows,  it  stands  out  from  the  gut  wall  and  finally  shows  the 
striations  indicative  of  the  presence  of  sporozoites;  after  rupture  of 


I 


% 
^   ^ 


c^jf^=5n 


Fig.  180. — ^ILemoprotbus  columb^.  la  to  3a,  Development  of  female  parasite  in 
blood  of  dove;  lb  to  3b,  Development  of  male  parasite  in  blood  of  dove;  4a,  4b, 
5b,  6  to  12,  Development  in  the  digestive  tube  of  the  fly  (Lynchia) ;  13  to  20, 
Development  of  the  parasite  inside  leucocytes  in  the  lung  of  the  dove.  (After 
Avagao.  MacNeal,  "Pathogenic  Microorganisms,"  published  by  P.  Blakiston's 
Sons  &  Co.) 


SPOROZOA 


763 


the  mature  cyst,  these  collect  in  large  numbers  in  the  salivary  glands 
and  ducts. 

The  life  history  of  the  parasite  is  seen  to  be  like  that  of  proteosoma 
and  malaria,  except  that  the  asexual  or  schizogenous  cycle  appears  to 
be  lacking. 

Proteosoma  ( Plasmodium)  prsecox. — This  parasite  is  a  typical  rep- 
resentative of  the  sporozoa,  and  is  interesting  historically,  since  it  was 
the  one  with  which  Ross  worked  in  1898,  when  he  first  demonstrated 
the  part  played  by  the 
mosquito  in  *'bird  ma- 
laria. '  * 

Grassi  and  Feletti  de- 
scribed the  parasite  in 
1890  under  the  name  of 
hemameha  precox.  It  is 
•widely  distributed  geo- 
graphically, and  is  com- 
mon in  the  blood  of  small 
birds,  sparrows,  robins  and 
larks.  It  can  be  propa- 
gated in  the  laboratory  in 
the  blood  of  canaries  with- 
out great  difficulty;  spar- 
rows, however,  do  not  long  survive  in  captivity  unless  kept  in  round 
glass  jars,  where  they  cannot  injure  themselves  by  dashing  against 
the  walls.  The  blood  for  examination  is  obtained  from  the  cephalic 
wing  vein,  close  to  the  body,  which  is  nicked  with  a  razor,  and  the 
blood  taken  up  in  a  capillary  glass  tube  containing  a  little  citrate 
solution.  To  inoculate  a  new  bird,  it  is  sufficient  to  inject  a  small 
quantity  of  citrated  blood  from  an  infected  canary  into  the  breast 
muscles  of  the  new  bird,  transferring  to  a  new  host  at  intervals  of 
a  month  or  less.  Because  it  is  not  difficult  to  keep  on  hand,  this  organ- 
ism may  be  used  for  class  study  in  localities  where  malarial  cases  are 
infrequent.  There  is  no  apparent  reason  for  placing  it  in  a  different 
genus  from  the  malarial  parasites. 

The  entire  asexual  cycle,  schizogony,  may  be  studied  in  the  periph- 
eral circulation,  as  in  quartan  malarial  fever. 

In  nature  it  is  transmitted  by  both  culex  and  stegomyia  {Aedes 
calopus),  and  its  development  is  briefly  as  follows:  The  bird  is  inocu- 
lated by  the  mosquito  with  spindle-shaped  young  forms  known  as 


A  B  C  D 

Fig.  181. — Proteosoma  precox  in  Blood  op 
Field  Lark.  A,  Young  parasite  in  blood  cell; 
B,  Half-grown  parasite  which  has  pushed  aside 
nucleus  of  blood  cell;  C,  Parasite  with  clump 
of  pigment  and  many  nuclei;  D,  Division  into 
many  merozoites.  (After  Doflein  and  Wasie- 
lewski.  MacNeal,  "Pathogenic  Microorgan- 
isms," published  by  P.  Blakiston's  Sons  &  Co.) 


764 


PATHOGENIC  PROTOZOA 


sporozoites.  These  possess  the  power  of  ameboid  motion,  and  rapidly 
penetrate  into  an  erythrocyte,  in  which  they  grow  quickly ;  they  con- 
stantly move  about  inside  the  cell  until  nearly  full  grown,  and  are 
during  this  stage  called  trophozoites.  The  substance  of  the  erythrocyte 
is  rapidly  consumed  by  the  parasite  and  a  dark  pigment,  melanin  or 


Fig.  182. — MroauT  of  Culex  Mosquito,  Covered  with  Oocysts  of  Proteosoma 
PRiECOX.  V,  Vasa  malpighii.  (After  Doflein  and  Ross.  MacNeal,  "Pathogenic 
Microorgaaisms,"  published  by  P.  Blakiston's  Sons  &  Co.) 

hemozoin,  is  formed  from  the  destroyed  hemaglobin.  The  mature 
parasite  divides  into  many  small  forms  called  merozoites,  and  these, 
when  freed  by  the  rupture  of  the  degenerated  erythrocyte,  escape  into 
the  blood  plasma,  and  if  not  phagocyted,  penetrate  other  erythrocytes 
and  repeat  the  asexual  or  schizogenous  cycle.  The  pigment  and  undi- 
vided portion  (restkorper)  of  the  cytoplasm  of  the  mother  cell 
accumulate  in  the  bone  marrow,  spleen  and  other  viscera. 


MALARIA 

This  is  one  of  the  most  common  and  widespread  of  preventable 
human  diseases,  and  in  some  localities  is  the  cause  of  a  greater  mor- 
tality and  morbidity  than  tuberculosis.  It  is  caused  by  one  or  more 
of  the  three  forms  of  the  malarial  Plasmodium.  As  a  rule  the  infec- 
tions are  simple,  yet  in  the  tropics  it  is  not  uncommon  to  find  two 
species  of  Plasmodia  in  the  same  patient,  and  this  condition  is  called 
a  mixed  infection. 

History. — The  disease  under  various  names,  as  chills  and  fever, 
Roman  fever,  Chagres  fever,  has  been  known  since  the  greatest  an- 
tiquity. The  cause  was  not  discovered  until  1880,  when  Laveran,  a 
French  military  surgeon  stationed  in  Algeria,  first  saw  the  organism 
and  described  it  as  the  cause  of  malaria.  He  saw  and  described  not 
only  the  pigmented  trophozoite,  but  also  the  crescentic  gametes  and 
flagellating  microgametocytes,  and,  because  of  the  activity  of  the 
flagella,  called  the  parasite  Oscillaria  malariw,  a  name  afterwards 


SPOROZOA  765 

given  up.  Later,  in  1885,  Celli  and  Marchifava  described  the  parasite 
with  greater  accuracy  and  named  it  Plasmodium  malarice,  a  poor 
name,  since  it  describes  merely  a  condition  assumed  by  some  fungi 
and  mycetozoa,  yet,  according  to  the  rules  of  zoological  nomenclature, 
it  must  stand.  In  the  same  year,  Golgi  described  the  quartan  parasite 
and  in  the  following  year  demonstrated  the  relation  of  the  various 
stages  of  the  life  cycle  of  the  tertian  parasite  to  the  temperature  curve. 

Even  in  antiquity  many  had  noted  the  curious  distribution  of 
malaria,  and  its  intimate  relation  to  swamps  and  marshy  places. 
Manson,  who  had  already  shown  the  role  played,  by  an  infected  mos- 
quito in  transmitting  filarial  disease,  in  1894,  suggested  that  the  epi- 
demiology of  the  disease  could  best  be  explained  on  the  hypothesis 
that  it  was  conveyed  by  the  bite  of  some  blood-sucking  insect,  probably 
the  mosquito. 

For  years  the  interpretation  of  the  flagella  was  a  subject  of  con- 
troversy. They  were  regarded  as  degeneration  products  by  some  and 
as  living  elements  by  others.  In  1897,  MacCallum,  working  with 
halteridium,  was  able  to  show  that  they  were,  in  fact,  spermatozoa,  as 
he  saw  them  penetrate  and  fertilize  the  macrogametes,  or  large 
spherical  forms  without  flagella. 

In  1897,  Ross,  of  the  British  Indian  Medical  Service,  described  the 
beginning  of  the  sporogenous  cycle  in  what  he  called  a  dapple-winged 
mosquito,  which  w^e  now  recognize  as  an  anopheline.  Following  out 
further  Hanson's  hypothesis,  he  was  able  the  same  year  after  long  and 
laborious  research  to  clear  up  the  method  of  transmission  of  bird 
malaria,  proteosoma,  an  analogous  disease.  Grassi  and  Bignami  and 
Bastianelli,  in  1898,  succeeded  in  demonstrating  the  complete  life  cycle 
of  the  human  form  of  malaria  in  the  anopheles  mosquito. 

Geographical  Distribution. — The  disease  is  found  in  a  belt  round 
the  world  extending  from  40  degrees  S.  latitude  to  60  degrees  N. ;  it  is, 
however,  not  equally  distributed  throughout  this  zone,  and  even  in  the 
tropics  there  are  many  malaria-free  areas,  principally  in  the  regions 
of  higher  altitudes,  since  the  special  home  of  malaria  is  in  the  low- 
lying,  swampy  and  torrid  coastal  districts  and  river  basins.  Islands 
at  a  distance  from  the  main  land  may  be  entirely  free.  Malaria 
reaches  its  maximum  intensity  in  the  tropics,  where  the  anopheline 
mosquitoes  breed  continuously  throughout  the  year,  and  new  infec- 
tions may  occur  at  any  time;  while  in  the  sub-tropics  and  temperate 
regions  it  is  a  seasonal  disease,  appearing  soon  after  the  onset  of  hot 
weather  with  its  new  crop  of  anophelines  and  continuing  until  the 


766 


PATHOGENIC  PROTOZOA 


first  cold  weather.  Relapses  continue  to  occur  throughout  the  winter 
season.  Modern  times  have  seen  it  disappear  from  many  regions 
where  it  was  formerly 'endemic,  because  of  increased  cultivation  of  the 
soil  and  better  surface  drainage,  as,  for  example,  in  England  and  the 
Ohio  river  valley. 

In  the  registration  area  of  the  United  States  there  were  1,565 
deaths  from  malaria  in  1913 ;  in  Italy,  up  to  1900,  the  average  number 
of  deaths  from  this  cause  annually  was  16,000.    One  cannot  obtain  a. 


o^^itS' 


w 


Fig.  183. —  Plasmodium  vivax. 
(Army  Med.  School  Collection, 
Washington,  D.  C.) 


Fig.  184. — Plasmodium  vivax.  (Gamete.) 
(Army  Med.  School  Collection,  Washing- 
ton, D.  C.) 


true  picture  of  the  importance  of  the  disease,  however,  from  mortality 
statistics,  since  it  is  not  often  fatal,  and  the  morbidity  is  out  of  pro- 
portion to  the  mortality.  In  many  villages,  where  it  is  endemic,  one- 
third  to  one-half  the  population  may  have  parasites  in  the  blood,  most 
of  them  without  clinical  symptoms,  yet  they  are  not  able  to  work  and 
the  children  remain  undeveloped  and  backward.  Much  of  the  illness 
attributed  to  hoolrworm  infection  is,  in  reality,  due  to  latent  malaria. 
The  parasites  belong  to  the  class  of  hemosporidia,  and  are  closely 
related  to  the  coccidia,  which  are  parasites  of  epithelial  cells,  while 
the  Plasmodia  are  parasitic  on  red-blood  cells.  There  are  tw^o  divisions 
of  the  life  cycle;  that  which  occurs  in  man,  the  endogenous,  asexual 
or  schizogenous,  and  that  which  occurs  in  the  mosquito,  the  exogenous, 
sexual  or  sporogenous;  for  this  reason  the  mosquito  is  the  definitive 
and  the  man  the  intermediate  host. 


SPOROZOA 


767 


There  are  three  well-recognized  forms  of  the  plasmodia,  (1)  Plas- 
modium vivax  (Grassi  and  Filetti),  causing  tertian  fever  (also  called 
''benign  tertian");  (2)  Plasmodium  malarice  (Laveran),  causing 
quartan  fever;  (3)  Plasmodium  falciparum  (immaculatum)  (Welch), 
causing  the  tropical  form  of  malaria,  the  so-called  astivo-autumnal 
or  subtertian.  Since  in  general  the  life  history  of  the  three  forms 
is  alike  they  will  be  considered  together  as  far  as  possible.     As  the 


^    \ 


Fig.  185. — Plasmodium  vivax,  an  Atypical 
Macrogametocyte.      Form  interpreted 

''  by  Schaudinn  as  undergoing  partheno- 
genesis. (Army  Med.  School  Collection, 
Washington,  D.  C.) 


Fig.  186.  —  Plasmodium  vivax. 
(Army  Med.  School  Collection, 
Washington,  D.  C.) 


details  of  development  cannot  be  made  out  easily  in  fresh  specimens, 
the  following  decription  applies  to  those  stained  with  some  form  of 
the  Romanowski  stain. 

Plasmodium  vivax. — The  parasite  of  tertian  fever  has  a  life  cycle 
lasting  forty-eight  hours  and  is  easily  recognized  only  when  full 
grown,  that  is,  twenty-four  to  forty-eight  hours  after  the  chill.  While 
a  diagnosis  may  be  made  on  younger  forms,  it  is  not  so  readily  made. 
As  its  name  implies,  the  Plasmodium  vivax  is  actively  ameboid, 
and  pseudopods  and  irregular  outlines  characterize  the  well-grown 
parasite ;  the  infected  erythrocyte  is  swollen,  often  to  twice  its  normal 
size,  the  hemoglobin  is  pale  and,  especially  in  spreads  in  which  Mar 
son's  stain  has  been  used,  it  is  so  much  paler  than  in  the  surrounding 
cells  that  the  infected  cell  stands  out  clearly.    The  part  of  the  cell  um 


76S  Pathogenic  protozoa 

occupied  by  the  parasite  is  stippled,  that  is,  dotted  with  reddish  gran- 
ules called  Schu,ffner's  dots,  and,  as  the  swollen  red  cell  and  Schuff- 
ner's  dots  are  found  in  no  other  form  of  malaria,  their  presence  is 
pathognomonic  of  tertian. 

The  youngest  form,  the  free  merozoite,  is  rarely  seen,  but  young 
comet-like  forms  composed  of  a  particle  of  red  chromatin  and  a  little 
blue  cytoplasm  may  readily  be  detected  at  the  height  of  the  fever; 
that  is,  a  few  hours  after  the  chill  and  sporulation.  The  round,  young 
schizont  as  it  grows  develops  early  a  central  vacuole  and  assumes 
the  shape  of  a  signet  ring,  the  red  chromatin  dot  being  the  stone.    This 


Fig.  187. — Plasmodium  vivax.    (Army  Med.  School  Collection,  Washington,  D.  C.) 

small  tertian  ring  grows  rapidly  as  the  fever  subsides,  and  at  the 
same  time  the  infected  cell  increases  in  size.  Twenty-four  hours  after 
the  chill  the  ring  has  grown  so  much  that  it  is  referred  to  as  the  large 
tertian  ring,  and  its  tendency  to  irregularities  of  shape  and  ameboid 
form  becomes  apparent,  and  fine  granules  of  pigment,  called  melanin 
or  hemozoin,  begin  to  be  visible.  After  thirty -six  hours  the  rings  will 
all  have  grown  into  large  ameboid  forms.  After  about  forty  hours  the 
parasite  occupies  almost  the  entire  cell  and  the  pigment  begins  to  col- 
lect in  masses  toward  the  center.  Soon  after  the  first  signs  of  seg- 
mentation appear,  which  becomes  more  and  more  distinct  until  fifteen 
'to  twenty  separate  segments  or  merozoites  are  seen,  each  composed 


SPOROZOA  769 

of  nucleus  and  cyptoplasm.  The  pigment  of  the  adult  parasite  and 
the  unused  portion  of  the  cytoplasm  are  cast  off  after  segmentation 
as  a  restkorper,  which  is  promptly  phagocyted  and  such  masses  ac- 
cumulate in  the  spleen,  bone  marrow  and  viscera.  With  rupture  of 
the  erythrocyte,  at  the  time  of  the  chill,  the  merozoites  are  set  free, 
and  if  not  phagocyted,  immediately  attack  new  erythrocytes  and  the 
asexual  or  schizogenous  cycle  is  repeated,  until  treatment  or  increas- 
ing immunity  halts  or  alters  the  cycle. 


Fig.  188. — Plasmodium  vivax.    (Army  Med.  School  Collection,  Washing '/>ii,  D.  C.) 

In  practice  it  is  not  unusal  to  find  parasites  of  different  ages  in 
the  same  film,  as  some  individuals  seem  to  develop  in  advance  of 
others;  in  this  case,  however,  there  will  not  be  much  difference  in 
their  appearance.  When  extreme  difference  of  age  is  noted  in  films  it 
is  probable  that  there  have  been  several  different  inoculations,  pro- 
ducing double  or  triple  infections  with  quotidian  or  irregular  fever 
curves,  and  such  cases  are  not  uncommon. 

As  all  the  forms  so  far  described  belong  to  the  schizogenous  cycle, 
they  may  be  called  schizonts,  or  trophozoites  of  the  schizogenous 
cycle.  The  sporogenous  cycle  begins  in  man  and  is  completed  in  the 
mosquito.  The  earliest  sexual  forms  noted  were  the  so-called  ' '  spheres, ' ' 
large  adult  parasites,  first  seen  in  wet  preparations,  which  did  not  seg- 


770  PATHOGlSNIC  PROTOZOA 

ment  with  the  schizonts.  They  are  now  called  gametes  and  after  the 
disease  has  lasted  some  time  are  found  in  films  made  at  all  stages  of 
the  fever;  that  is,  they  are  incapable  of  further  development  un- 
til taken  into  the  stomach  of  the  mosquito.  The  possibility  of 
parthenogenesis  will  be  referred  to  latir.  In  appearance  they  are 
round  or  oval,  and  in  this  fever  may  be  twice  the  size  of  the 
red  cell.  As  a  rule  a  narrow  margin  of  red  cell  is  visible  after  Ro- 
manowski  stains,  although  the  gamete  may  lie  free  in  the  plasma. 
Unlike  the  schizonts,  the  gametes  have  the  pigment  uniformly  dis- 
tributed throughout  the  body  and  there  is  no  indication  of  segmen- 
tation. The  young  sporonts  are  distinguished  from  schizonts  by  the 
absence  of  the  vacuole,  and,  when  a  little  older,  by  a  larger  amount 
of  hemozoin. 

Plasmodium  malariae. — The  quartan  parasite  has  a  life  cycle  of 
seventy -two  hours,  or  twenty-four  hours  longer  than  the  tertian,  and 


r 


^ 


Fig,  189. — Plasmodium  malaria.  (Army     Fig.  190. — Plasmodium  malaria.  (Army 
Med.  School  Collection,  Washington,  Med.  School  Collection,  Washington^ 

D.  C.)  D.  C.) 

the  paroxysms  come  on  every  third  day,  or,  according  to  the  Italian 
method  of  reckoning  time,  on  the  fourth  day.  The  young  rings  of  the 
Plasmodium  malarice  are  indistinguishable  from  young  tertian  rings, 
but  the  diagnosis  may  be  made  on  older  forms.  The  bleaching,  enlarge- 
ment and  stippling  of  the  erythrocyte  characteristic  of  tertian  is  never 
found  in  quartan  fever,  the  infected  erythrocyte  being  almost  normal 
in  appearance.  The  well-grown  quartan  parasite  does  not  show 
ameboic  changes  but  assumes  a  band  form,  more  or  less  wide,  stretch- 
ing across  the  red  cell  from  b(  rder  to  border ;  with  increasing  age  the 


SPOROZbA 


771 


band  widens  until  the  parasite  is  nearly  square  and  the  hemozoin  ac- 
cumulates toward  the  center.  Segmentation  gives  rise  to  almost  sym- 
metrical "daisy"  forms,  showing  six  to  eight  or,  rarely,  fourteen  mero- 
zoites.  Parasites  of  different  ages  may  be  found,  as  in  tertian,  and 
it  is  characteristic  of  quartan  fever  that  examples  of  all  stages  of 
the  life  cycle  may  be  found  at  the  proper  time  in  the  peripheral  cir- 
culation. Gametes  differ  from  tertian  mainly  in  size,  since  they  are 
never  larger  than  the  normal  erythrocyte  until  after  the  latter  has 
ruptured,  but  when  free  in  the  plasma  it  is  practically  impossible  to 
distinguish  them  from  tertians. 


Fig.  191.  —  Plasmodium  malari^e. 
(Army  Med.  School  Collection,  Wash- 
ington, D.  C.) 


Fig.  192.  —  Plasmodium  falciparum. 
(X  1500.)  (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 


Plasmodium  falciparum. — The  parasite  of  aestivo-autumnal  fever, 
Plasmodium  falciparum,  differs  considerably  from  the  two  forms  al- 
ready described;  the  life  cycle  varies  between  twenty-four  and  forty- 
eight  hours,  and,  at  least  in  new  itifections,  only  ring  forms  are 
jEound  in  the  peripheral  blood,  although  at  a  later  stage  crescentic 
gametes  may  be  present.  The  youngest  aestivo-autumnal  rings,  found 
at  the  height  of  the  fever,  are  more  delicate  than  the  young  tertians. 
As  the  temperature  falls  the  rings  increase  in  size,  but  without 
change  of  form ;  the  growth  is  not  uniform,  but  occurs  as  a  thick  cres- 
centic swelling  on  the  convex  surface  of  the  ring,  and  occasionally 
more  than  one  such  swelling  is  present.  The  large  aestivo-autumnal 
ring,  found  after  the  febrile  paroxysm  has  passed,  occupies  one-third 
to  one-half  the  red  cell,  which  is  never  swollen  nor  stippled,  as  in  ter- 
tian, and  the  parasite  is  never  band-like,  as  in  quartan.     Segmenting 


772 


PATHOGENIC  PROTOZOA 


parasites  are  almost  never  seen  in  the  peripheral  blood  in  aestivo- 
autumnal  fever,  though  in  tertian  they  are  common  and  in  quartan 
numerous.  If,  however,  films  are  prepared  at  autopsy  from  the 
spleen,  liver,  bone  marrow  and  brain,  enormous  numbers  of  segment- 
ing forms,  together  with  all  other  stages  of  the  parasite,  may  be  found. 
The  full  grown  segmenter  occupies  one-third  to  one-half  the  cell  and 
shows  a  collection  of  hemozoin  in  large  blocks  in  the  center,  The 
merozoites  vary  in  number  from  eight  to  twenty -five.  In  addition 
to  the  small  and  large  rings  the  peripheral  blood  shows,  after  the  fever 
has  lasted  sufficiently  long,  the  sexual  forms  or  gametes.  The  infected 
erythrocyte  is  never  stippled  nor  swollen,  but,  on  the  contrary,  may 
appear  shrunken.  Both  the  micro-  and  macrogametocytes  in  sestivo- 
autumnal  fever  are  crescentic  in  shape,  their  length  being  about  one 

and  one-half  and  the  width  about 
one-half  that  of  an  erythrocyte; 
the  pigment  is  collected  toward  the 
center,  which  is  rather  paler  in 
stained  specimens  than  the  poles. 
At  first  sight  the  gametes  appear 
to  lie  free  in  the  plasma,  yet  in 
stained  specimens  a  rim  or  rib  of 
the  pale  red  cell  may  be  seen  on 
the  concave  side.  When  liberated 
from  the  erythrocyte  the  gamete 
becomes  first  spindle-shaped  and 
finally  oval  or  round.  The  male 
crescent  is  short  and  broad,  and 
the  female  relatively  long  and 
slender. 
The  Finer  Structure  of  the  Plasmodia. — The  finer  details,  w'nich 
are  only  hinted  at  in  fresh  specimens  and  in  those  stained  with  Man- 
son's  stain,  can  be  studied  to  advantage  in  those  stained  with  some 
one  of  the  many  modifications  of  the  Romanowski  stain,  such  as  that 
of  Wright,  Hastings,  MacNeal  or  Giemsa. 

The  tertian  parasite,  which  lies  in  a  red  cell,  may  be  seen  to  be 
divided  into  a  blue  cytoplasm  and  a  brilliant  red  nucleus,  and  it 
would  be  well  for  the  novice  to  remember  that  these  three  conditions 
m.ust  be  satisfied  before  the  diagnosis  of  malaria  can  be  made;  the 
principal  stumbling-block  is  the  blood  platelet,  often  found  overlying 
a  red  cell,  but  it,  although  possessing  a  ragged  blue  cytoplasm,  has  al- 


FiG.  193. — Plasmodium  falciparum 
(X  1500.)  (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 


SPOROZOA 


773 


ways  a  relatively  large  purple  nucleus.  The  chromatin  of  the  young 
rings  is  usually  present  as  a  single  dot,  but  two  such  dots  are  frequently 
seen.  In  older  forms  of  the  tertian  and  quartan  parasites  the  various 
changes  found  in  mitosis  may  be  followed  in  the  nucleus.  The  whole 
schizogenous  cycle  may  be  followed  by  taking  blood  smears  from  a 
single  case  of  malaria  at  intervals  of  three  or  four  hours  for  forty- 
eight  hours  for  tertian  and  aestivo-autumnal,  and  for  seventy-two 
hours  for  quartan. 


Fig.  194. — Plasmodium  falciparum,  Male  Crescent.     (Army  Med.  School  Col- 
lection, Washington,  D.  C.) 

Two  forms  of  sporonts  or  gametes  may  be  seen ;  in  one  the  quantity 
of  chromatin  is  large  and  the  cytoplasm  pale  blue,  while  in  the  other 
the  reverse  is  found,  the  nuclear  chromatin  is  comparatively  small  in 
quantity  and  the  cytoplasm,  being  rich  in  nutrient  material,  stains 
deeply.  The  first  form,  with  abundant  chromatin,  is  the  male,  or 
microgametocyte,  and  the  latter  the  female,  or  macrogametocyte.  The 
differentiation  between  schizont  and  sporont  may  be  made  while  the 
parasites  are  still  quite  young;  since  the  schizont  is  characterized  by 
the  presence  of  a  nutrient  vacuole,  and  the  sporont,  of  equal  age,  while 


774  -  PATHOGENIC  PROTOZOA 

lacking  the  vacuole,  shows  a  greater  amount  of  hemozoin,  which  is 
never  concentrated  in  the.  center  of  the  parasite  but  is  scattered 
equally  throughout  the  body.  The  cytoplasm  of  the  sporont  is  less 
fluid  than  that  of  the  schizont  and  shows  no  tendency  to  ameboid 
motion.  The  chromatin  is  relatively  large  in  amount  and,  although 
broken  up  more  or  less  into  granules  and  threads,  shows  no  real  ten- 
dency to  segment  or  disperse,  but  remains  a  compact  mass. 

The  quartan  parasite,  when  stained  with  Wright's  or  a  similar 
preparation,  shows  quite  regular  and  symmetrical  segmentation,  usu- 
ally into  eight  merozoites.  The  distinction  between  schizont  and 
sporont  and  between  male  and  female  gametes  may  be  made  on  the 
same  grounds  as  in  tertian. 

In  aestivo-autumnal  fever  the  chromatin  dot  in  the  young  ring  is 
often  doubled,  or  even  trebled,  and  in  general  is  large  and  stains 
brilliantly.  The  adult  and  half -grown  gametes  may  be  differentiated 
into  male  and  female  by  the  criteria  already  given. 

The  Examination  of  Fresh  Blood. — Directions  have  already  been 
given  (Chap.  LIX)  for  making  wet  preparations  and  if,  by  ringing 
the  cover-glass  with  vaseline,  drying  be  prevented,  the  preparations 
will  keep  and  may  be  studied  for  hours.  In  tertian  fever  the  young 
ring  forms  are  at  first  difficult  to  detect,  unless  the  amount  of  light 
going  through  the  microscope  be  cut  down  to  the  minimum.  As  the 
parasite  grows  older,  an  increasing  number  of  hemozoin  granules  ap- 
pear, and  since  they  are  in  constant  motion  the  parasite  is  readily  de- 
tected. Its  cytoplasm  is  delicate,  and  with  very  young  parasites  is 
difficult  to  distinguish  from  the  red  cell  itself ;  older  parasites,  however, 
develop  pseudopods,  which  are  constantly  projected  and  retracted,  and 
the  entire  organism  shows  active  movements,  rendering  it  easy  to  see. 
The  pigment  continues  to  increase,  and  in  the  gametes  is  abundant 
and  in  constant  motion;  the  gametes,  however,  fail  to  show  any  ame- 
boid changes,  and  the  protoplasm  is  stiff  and  rigid  with  a  regular,  un- 
broken margin.  At  times  a  clear  refractile  spot  is  seen,  which  is  the 
nucleus.  The  infected  erythrocyte  is  pale  and  swollen.  Even  in  un- 
stained preparations  the  sexes  may  be  distinguished ;  the  microgameto- 
cyte  is  about  the  size  of  a  red  cell,  the  cytoplasm  is  hyaline,  and  after 
the  preparation  has  been  made  ten  to  twenty  minutes  the  flagella,  or 
microgametes, .  may  be  seen  thrashing  about  in  the  parasite.  After 
repeated  attempts  four  to  eight  microgametes  rupture  the  cell  and 
emerge.  The  macrogamete  is  larger  than  a  red  cell  and  is  finely 
granular  and  no  exflaggelation  of  microgametes  occurs. 


SPOROZOA  775 

In  quartan  malaria  the  differences  already  described  in  stained 
blood  may  be  easily  followed. 

In  agstivo-autumnal  fever  the  diagnosis  with  fresh  blood  is  much 
more  difficult  in  new  infections  because  of  the  relative  scarcity  of  the 
parasites  in  the  peripheral  blood  and  the  exceedingly  small  size  of  the 
young  rings,  the  absence  of  hemozoin  in  them,  and  the  very  slight 
ameboid  motion.  The  older  rings  are  larger,  contain  some  pigment 
and  are  more  easily  seen.  The  infected  erythrocyte  is  never  pale  nor 
swollen,  but,  on  the  contrary,  may  be  shrunken  and  brassy  in  color. 
The  crescentic  gametes  are  readily  detected,  and  the  sexes  may  be 
differentiated  by  their  shape  and  the  hyaline  or  granular  character 
of  the  cytoplasm. 

Incubation  Period  of  the  Malarial  Fevers. — Two  methods  have 
been  used  to  determine  this  point — the  injection  of  infected  malarial 
blood,  and  biting  experiments  with  infected  anophelines.  By  the  first 
method  the  incubation  period  was  eighteen  days  (the  longest)  for 
quartan,  three  days  (the  shortest)  for  aestivo-autumnal,  and  ten  days 
for  tertian.  By  the  second  method  aestivo-autumnal  was  nine  to 
twelve  days,  and  tertian  fourteen  to  nineteen  days.  Since  aestivo- 
autumnal  is  the  only  parasite  which  can  complete  its  cycle  in  twenty- 
four  hours,  the  short  incubation  period  is  easily  understood;  on  the 
other  hand,  the  long  life  cycle  of  quartan,  seventy -two  hours,  explains 
its  slower  development. 

Clinical  descriptions  of  the  malarial  fevers  may  be  found  in  the 
standard  text-books  on  medicine,  and  it  is  only  necessary  here  to  refer 
briefly  to  the  various  forms  found  in  practice.  The  classical  malarial 
fever  consists  of  a  series  of  paroxysms,  following  one  another  with  a 
definite  periodicy,  daily,  every  other  day,  or  every  third  day.  Each 
paroxysm  is  ushered  in  by  a  pronounced  chill,  which  is  sometimes  pre- 
ceded by  malaise,  headache  and  lassitude.  The  chill  lasts  from  ten 
minutes  to  an  hour  or  more,  and  the  patient  wraps  himself  up  in 
heavy  blankets.  During  the  chill  the  temperature  begins  to  rise  and 
within  a  few  hours  reaches  its  high  point,  103°  to  106°,  and  then 
falls  slowly  to  normal  during  the  next  few  hours.  The  decline  of  the 
fever  is  accompanied  by  a  profuse  perspiration.  Successive  parox- 
ysms may  occur  at  exactly  the  same  hour  of  the  day,  or  may  antici- 
pate, fehris  anteponens,  or  be  delayed  an  hour  or  more,  fehris  post- 
ponens.  The  sequence  of  events,  therefore,  in  a  typical  malarial 
paroxysm  is  malaise,  chill,  "fever  and  sweat,  followed  by  a  period  of 
apparent  well-being. 


776  PATHOGENIC  PROTOZOA 

1.  Tertian  malaria  is  distinguished  by  a  chill  and  fever  occurring 
every  other  day,  the  patient  feeling  quite  well  on  fever-free  days,  A 
double  tertian  infection  occurs  not  infrequently,  giving  a  daily,  or 
quotidian,  chill  and  fever  with  no  free  day. 

2.  Quartan  fever,  which  is  relatively  rare,  gives  a  chill  and  fever 
every  third  day,  with  two  fever-free  days.  In  this  disease  also  there 
may  be  double  or  even  triple  infections,  giving  a  quotidian  or  irreg- 
ular type  of  fever. 

3.  ^stivo-autumnal  fever  (subtertian,  or  malignant  tertian)  shows 
an  irregular  temperature  curve,  the  cycle  varying  from  twenty-four 
to  forty-eight  hours.  By  some  authors  the  disease  is  divided  into 
two  forms,  quotidian  and  tertian;  and  as  multiple  infections  are  com- 
mon the  resulting  fever  curve  may  be  irregular  or  continuous  and  the 
chill  entirely  absent.  In  contrast  to  the  regular  intermittency  of  ter- 
tian and  quartan  this  form  is  often  remittent,  the  temperature  curve 
never  dropping  to  normal. 

4.  Mixed  infections  with  any  two  of  the  above  fevers  are  often 
found  in  bad  malarial  regions  in  the  tropics. 

5.  Latent  malaria  is  also  not  infrequent,  in  which  the  patient,  hav- 
ing no  symptoms  of  the  disease,  consults  a  physician  for  some  other 
reason. 

6.  The  carrier  state  is  found  among  natives  or  persons  long  resi- 
dent in  malarial  regions,  and,  aside  from  the  presence  of  a  large  spleen 
and  some  secondary  anemia,  may  present  no  symptoms.  It  is  particu- 
larly common  among  native  children,  tramps  and  vagabonds.  It  is  not 
uncommon  to  find  fifty  to  one  hundred  per  cent  of  the  children  in  a 
native  village  harboring  the  parasite. 

THE  DEVELOPMENT  OF  THE  HUMAN  MALARIAL  PARASITE 
IN  THE  MOSQUITO 

{Sexual  half  of  the  life  cycle ,  Sporogonie) 
For  this  stage  to  be  successful  the  mosquito  must  bite  a  malarial 
patient  with  gametes  in  his  blood,  for  if  the  patient  be  one  in  the  first 
stage  of  the  disease,  with  only  schizonts  in  his  blood,  no  infection  of 
the  mosquito  will  take  place,  since  the  schizonts  all  perish  in  its 
stomach.  On  the  contrary,  if  the  mosquito  takes  blood  from  a  per- 
son who  has  been  ill  with  malaria  for  some  time,  or  from  an  appar- 
ently healthy  carrier,  the  schizonts  die  as  usual,  but  the  gametes  find 
in  the  mosquito  stomach  for  the  first  time  conditions  suitable  for 
their  further  development. 


SPOROZOA  777 

The  various  stages  may  be  studied  by  causing  suitable  species  of 
anophelines  to  bite  persons  with  many  gametes  in  their  blood,  and 
then  dissecting  the  stomach  and  observing  the  changes  which  take 
place  there.  The  development  is  visible  in  unstained  specimens  with 
high,  dry  lenses.  Since  there  is  no  essential  dift'erence  in  the  develop- 
ment of  the  three  forms  of  malaria  in  the  mosquito,  they  will  be  con- 
sidered together.  The  first  stage  has  already  been  described  in  dis- 
cussing the  appearance  and  behavior  of  gametes  in  fresh  blood.  In 
the  mosquito  the  process  may  be  followed  further;  the  macrogamete, 
freed  from  its  enveloping  red  cell,  projects  a  little  mound  on  its  sur- 
face, and  this  apparently  attracts  the  microgametes  to  its  neighbor- 
hood. Into  this  microphyle  one,  but  never  more,  of  the  flagella  pene- 
trates, following  which  the  mound  is  instantly  retracted.  The  fer- 
tilized macrogamete,  now  called  a  "zygote,"  soon  develops  the  powei 
<of  vermicular  motion,  then  being  called  an  ookinet,  and  travels  to 
the  wall  of  the  stomach,  and,  like  the  coccodia,  penetrates  an  epithelial 
cell  and  there  encysts,  making  the  oocyst.  This  grows  rapidly  and 
soon  escapes  from  its  host  cell  and  comes  to  lie  in  the  outer  layers 
of  the  stomach  wall,  and  as  it  grows  projects  into  the  body  cavity  of 
the  mosquito.  The  nucleus  divides  repeatedly,  always  accompanied 
by  some  of  the  cytoplasm,  forming  numerous  sporoblasts,  and  these, 
in  turn,  subdivide  into  innumerable  sporozoites;  these  last  escape, 
with  the  rupture  of  the  oocyst,  into  the  body  cavity.  From  there 
they  pass  to  all  parts  of  the  mosquito,  but  especially,  perhaps  be- 
cause of  chemotaxis,  to  the  salivary  glands  and  ducts,  and  when 
next  the  mosquito  bites  a  warm-blooded  host  the  sporozoites  enter  the 
blood  stream  and  start  life  anew. 

Cultivation  of  the  Malarial  Parasites  in  Vitro. — Bass  and  Johns  in 
1911  announced  the  cultivation  of  a  few  generations  of  Plasmodium 
vivax  in  vitro  under  strict  anaerobic  conditions.  Ten  c.c.  or  more  of 
blood  from  a  malarial  patient  is  defibrinated  and  distributed  in  small 
test  tubes  in  one  c.c.  quantities  and  to  it  is  added  one  per  cent  of  a 
fifty  per  cent  solution  of  glucose.  The  red  cells  settle  so  that  they  are 
covered  with  one-half  cm.  of  serum ;  the  parasites  grow  in  a  thin  layer 
near  the  top  of  the  cell  mass ;  beneath  this  they  die,  or  are  phagocyted. 
The  optimum  temperature  is  19°  to  40°  C. 

Bass  states  that  he  has  cultivated  all  three  species  of  Plasmodia 
by  destroying  the  complement  by  heating  one-quarter  to  one-half  hour 
at  40°  C.  Under  strict  anaerobiasis  it  was  possible  to  transfer  the 
cultures  and  to  keep  them  alive  for  twenty  days. 


778  PATHOGENIC  PROTOZOA 

Transmission  is  solely  by  various  species  of  anopheles  mosquitoes. 
Although  there  are  fifty  or  more  recognized  species,  only  sixteen  have 
been  proved  malarial  carriers.  The  more  important  are  Anopheles 
quadrimaculatus  in  the  United  States,  Anopheles  alhimanus  in  the 
American  tropics,  Anopheles  maculipennis  in  Europe,  Anopheles  sen- 
ensis  in  India,  and  Anopheles  costalis  in  Africa. 

Description  of  the  Mosquito. — It  is  impractical  to  give  more  than 
a  hasty  description  of  mosquitoes,  and  the  reader  is  referred  to  larger 
works  on  the  subject  (Howard,  Dyer  and  Knab;  Theobald  or  '^ Medi- 
cal Entomology,"  Patton  and  Cragg,  London,  Madras  and  Calcutta, 
1913).  It  may  be  noted,  however,  that  the  Culicidce,  or  mosquitoes, 
belong  to  the  Diptera,  or  two-winged  insects.  They  pass  through  four 
distinct  stages  in  their  development,  the  egg,  larval,  pupal  and  adult 
or  imago  stage.  The  eggs  are  invariably  laid  in  water  where,  if  the 
temperature  is  warm,  they  hatch  in  one  to  four  days.  Anopheles 
eggs  are  single  and  oval,  supported  on  the  surface  of  the  water  by 
ornamental  air  cells;  those  of  culex  are  cemented  together  when  laid 
in  raft-like  masses. 

The  larvae  are  aquatic  and  die  quickly  out  of  water.  They  are 
both  bottom  and  top  feeders,  eat  voraciously,  consuming  algae  and 
other  vegetable  matter,  and  some  varieties  are  cannibalistic.  The 
larvae  are  provided  with  a  breathing  tube  or  respiratory  siphon  pro- 
jecting upward  from  the  dorsal  surface  at  the  caudal  end,  and  in 
breathing  this  is  thrust  upward  to  the  surface  of  the  water  and  the 
larva  hangs  suspended  from  the  surface  film.  In  the  anophelines  the 
breathing  tube  is  short  and  its  angle  with  the  body  is  such  that  the 
larva  lies  parallel  with  the  surface;  with  culex  and  other  genera  the 
body  lies  at  an  angle  with  the  surface.  The  larval  stage  lasts  about 
six  to  fourteen  days,  depending  upon  the  temperature  and  food  sup- 
ply, and  is  followed  by  the  pupal  stage,  during  which  no  feeding 
occurs;  the  pupa,  however,  needs  air  and  is  provided  with  a  short 
respiratory  tube  at  each  side  of  the  head.  The  habit  of  coming  to  the 
surface  of  the  water  to  obtain  air,  which  obtains  in  all  mosquitoes 
except  the  Mansonia,  gives  a  point  of  attack  in  combating  them,  since  a 
layer  of  mineral  oil  on  the  surface  of  the  water  occludes  the  respira- 
tory siphon  and  so  kills  them. 

The  adult,  or  imago,  emerges  from  the  pupa  when  the  latter  is  one 
to  three  days  old;  the  pupal  case  ruptures  along  its  dorsum  and  the 
emerging  imago  rests  on  the  floating  pupal  case  until  its  wings  are 
dry.     Since  this  is  a  critical  stage  in  its  life  history  and  demands 


SPOROZOA 


779 


a 


A 


I 


Fig.  195. — Comparison  of  Culex  (left)  and  Anopheles  (right).  Eggs;  larvae  (note 
position);  position  of  insects  at  rest;  wings;  heads  showing  antennae  and  palpi. 
(After  KoUe  and  Hetsch.    Jordan,  "General  Bacteriology,"  Saunders.) 


780  PATHOGENIC  PROTOZOA  ^ 

quiet  water,  it  is  evident  that  the  least  wave  action  is  fatal  to  the 
mosquito. 

The  nature  of  the  breeding  place  is  characteristic,  to  a  certain 
extent,  of  each  genus  of  culicidw;  stegomyia  {cedes  calopus),  the  car- 
rier of  yellow  fever,  for  example,  is  strictly  domestic  and  breeds  in 
water  jars,  tin  cans,  old  beer  bottles  and  other  artificial  collections 
of  water ;  wyeomyia  breeds  exclusively  in  the  fluid  at  the  base  of  the 
leaves  of  air  plants;  {hromeliads)  the  anophelines,  while  domestic  to 
the  extent  that  they  live  near  human  habitations,  require  natural  col- 
lections of  water  for  breeding  places,  such  as  sheltered  spots  along  the 
overgrown  banks  of  streams,  temporary  puddles  and  even  in  water  in 
the  footprints  of  man  and  animals. 

A  proper  classification  of  mosquitoes  requires  considerable  train- 
ing, but  the  following  points  will  suffice  to  separate  the  anophelines 
from  other  mosquitoes.  On  either  side  of  the  proboscis,  as  seen  under 
a  hand  lens,  are  two  pairs  of  organs;  next  to  the  proboscis  are  the 
palpi,  and  outside  of  these  the  antennae ;  the  latter  serve  to  distinguish 
the  sexes,  the  antennae  of  the  male  being  heavily  ornamented  with  a 
bushy,  hairy  investment  (plumose)  ;  the  female  antennae,  on  the  con- 
trary, are  provided  with  relatively  few,  short  hairs,  arranged  in  rings 
at  the  joints  (pilose).  In  the  anophelines  the  palpi  in  both  sexes  are 
long,  at  least  as  long  as  the  proboscis,  while  in  all  other  mosquitoes 
they  are  short  in  the  female  or  in  both  sexes.  This  is  the  principal 
differential  point.  The  wing  markings  are  of  some  help,  since  ano- 
pheline  wings  are  almost  always  spotted.  Quite  characteristic  also  is 
the  position  assumed  by  both  genera  while  at  rest;  among  the  ano- 
phelines the  head,  thorax  and  abdomen  are  all  in  a  straight  line  and 
the  insect  makes  an  angle  with  the  surface  upon  which  it  rests ;  while 
the  culicidae  are  hump-backed,  the  thorax  and  head  are  bent  on  the 
abdomen  so  that  the  latter  lies  parallel  to  the  surface.  By  these  char- 
acteristics it  is  easy  to  identify  a  mosquito  as  an  anopheline,  but  the 
further  classification  into  species  is  less  simple,  and  works  on  ento- 
mology must  be  consulted. 

The  life  history  of  the  anophelines  is  not  yet  completely  known ; 
they  fly  and  bite  at  dusk  and  dawn  and  during  the  night,  and  thus 
differ  from  stegomyia  and  most  other  culicidae,  which  are  day-time 
biters.  Their  breeding  places  have  already  been  described ;  of  special 
importance  are  the  temporary  collections  of  water  in  which  the  larvae, 
unhampered  by  their  natural  enemies,  quickly  reach  maturity  in  large 
numbers.    With  abundance  of  food  and  warm  weather,  the  larvae  may 


SPOROZOA  781 

require  no  more  than  ten  days  to  complete  their  three  molts.  The 
female  alone  sucks  blood,  the  male  living  on  fruit  and  vegetable  juices. 
Egg-laying  does  not  take  place  until  after  a  meal  of  blood,  and  it  is 
possible  that  the  female  journeys  fairly  long  distances  to  obtain  this 
food,  and  that  the  first  flight,  from  breeding  place  to  human  habita- 
tions, may  be  longer  than  subsequent  flights.  In  general  the  flight  is 
short,  not  over  three  hundred  yards,  and  Gorgas  found,  in  Panama, 
that  a  clearing  of  that  width  about  houses  gave  ample  protection. 

The  incubation  period  of  the  parasite  in  the  mosquito  is  about 
twelve  days,  after  which  the  insect  remains  a  carrier  during  the  rest 
of  its  life,  and  as  the  health  of  the  mosquito  is  unaffected,  this  may 
be  for  two  months  or  more. 

Epidemiology. — Since  malaria  is  conveyed  solely  by  the  bite  of.  an 
infected  anopheline,  the  epidemiology  is  comparatively  simple.  In 
practice,  nevertheless,  the  prevention  of  the  disease  is  extremely  diffi- 
cult ;  but  it  is  the  same  for  all  forms  of  malaria.  It  must  be  attacked 
from  all  possible  angles  and  the  following  are  the  main  points  to  be 
observed : 

1.  Screened  houses  afford,  perhaps,  the  simplest  form  of  protec- 
tion, and  in  beginning  work  in  a  new,  badly  infected  place,  should 
be  the  first  thing  provided,  since  they  afford  a  place  of  security  in  an 
otherwise  dangerous  area,  where  the  workers  may  take  refuge  until 
the  situation  is  under  control.  This  method  alone  has  given  magnifi- 
cent results  in  Italy  (Celli)  since  it  was  first  used  experimentally  by 
Sambon  and  Low  in  the  Eoman  Campagna.  The  screens,  to  be  dur- 
able, must  be  of  bronze  and  not  iron,  and  of  a  fine  mesh  (20  strands 
to  the  inch),  and  should  be  placed,  not  on  windows  and  doors,  but  on 
the  outside  of  porches  and  balconies;  doorways  should  have  screened 
vestibules. 

In  default  of  metallic  house  screens,  bed  nets  may  be  used,  al- 
though they  are  not  very  satisfactory,  since  one  must  retire  at  dusk 
to  be  protected.  In  default  of  both  screens  and  bed  nets,  something 
may  be  accomplished,  temporarily,  by  daily  mosquito  catching,  and 
in  Panama  the  method  has  given  remarkable  results.  A  native, 
armed  with  a  small  acetylene  lantern  and  a  few  catching  bottles, 
soon  becomes  expert,  and  can  capture  each  day  all  the  mosquitoes  in 
a  number  of  dwellings.  In  this  way  very  few  anophelines  escape  cap- 
ture long  enough  to  become  infective  for  man.  Chloroform  catching 
bottles  are  easily  prepared  by  packing  a  half  ounce  of  small  rubber 
bands,  cut  up  finely,  into  the  bottom,  and  pouring  in  as  much  chloro- 


782  PATHOGENIC  PROTOZOA 

form  as  the  rubber  will  absorb  and  covering  it  over  with  dry  blotting 
paper.  The  wide-mouthed  catching  bottle  is  uncorked  and  inverted 
over  the  resting  mosquito,  which  is  killed  by  the  chloroform.  A  con- 
venient trap  bottle  has  been  described  by  La  Prince.  Below  the  cork 
is  placed  a  funnel  trap,  making  it  possible  to  pass  from  one  spot  to 
another  without  waiting  for  the  chloroform  to  act  upon  the  mosquito. 

2.  The  second  measure  is  the  prevention  of  mosquito  breeding; 
this  is  a  large  but  not  a  hopeless  undertaking,  if  carried  out  intelli- 
gently. In  the  first  place,  it  is  to  be  remembered  that  comparatively 
few  mosquitos  are  disease  carriers,  and  that  measures  need  be  directed 
against  them  only.  It  was  first  shown,  for  example,  by  Gorgas,  in 
Havana,  that  stegomyia  could  be  practically  exterminated  by  doing 
away  with  water  in  artificial  containers  in  the  neighborhood  of  dwell- 
ings ;  this  so  reduced  their  numbers  that  when  a  case  of  yellow  fever 
was  introduced  into  a  community  the  disease  would  not  spread.  In 
the  same  way  malaria-carrying  mosquitoes  may  be  fought,  without 
regard  to  the  presence  of  non-disease  carriers.  An  accurate  mosquito 
survey  is  first  made,  both  by  examination  of  the  catch  of  adults  and 
by  a  hunt  for  larvae  in  collections  of  water  throughout  the  district. 
A  puddle  or  stream  is  examined  by  dipping  with  a  white  saucer  or  a 
long-handled  dipper,  along  the  margins;  after  a  little  practice  it  is 
possible  to  obtain  any  larvae  which  may  be  present,  and  to  decide  by 
their  appearance  and  behavior  whether  they  are  anophelines.  It  is, 
of  course,  unnecessary  to  waste  time  and  money  destroying  collections 
of  water  free  from  anophelines.  When  the  breeding  places  have  been 
located,  they  may  be  destroyed  by  the  use  of  oil  or  larvacide,  by  drain- 
ing or  by  filling. 

The  use  of  oil  and  larvacides,  while  usually  a  temporary  measure, 
is,  nevertheless,  of  great  importance;  any  light  fuel  oil  may  be  used 
by  spraying,  by  mopping  the  sides  of  ditches  or  margins  of  ponds,  or 
by  a  drip  barrel  at  the  head  of  a  water  course.  Since  in  the  malarial 
season  the  mosquito  develops  rapidly  the  oil  must  be  applied  regu- 
larly once  a  week. 

Wherever  possible,  the  swampy  areas  and  pools  must  be  drained, 
and  for  this  purpose  both  agricultural  tile  and  open  ditches  may  be 
used;  in  the  tropics  it  is  necessary  to  line  the  latter  with  concrete  to 
prevent  overgrowth  by  rank  vegetation  and  to  protect  the  banks 
against  caving.  Where,  because  of  the  lay  of  the  land,  drainage  is 
impracticable  the  area  may  be  filled,  or  sometimes  flooded,  or  irrigated 
with  sea  water,  in  which  most  anophelines  do  not  survive.    It  is  better 


SPOROZOA  783 

in  the  long  run  to  begin  in  the  center  of  the  settlement,  with  perma- 
nent improvements,  working  outwards  as  money  becomes  available, 
and  not  to  depend  on  oiling  except  as  a  temporary  measure. 

3.  The  infection  is  kept  alive  in  a  community  by  human  carriers, 
and  these  are  especially  common  among  natives  and  the  poor  and 
ignorant.  Newcomers  should  not  live  within  five  hundred  yards  of  the 
dwellings  of  these  classes,  as  infected  anophelines  can  easily  travel 
shorter  distances.  Only  when  it  is  possible  to  protect  the  natives  also 
by  these  measures  is  it  safe  to  live  among  them ;  and  that  this  is  pos- 
sible has  been  shown  many  times,  particularly  in  Panama. 

4.  The  proper  treatment  and  cure  of  all  cases  will  not  only  pre- 
vent relapses  and  the  carrier  state  (malarial  cachexia),  but  is  one  of 
the  most  important  means  of  exterminating  the  disease,  since  every 
neglected  case  becomes  a  focus  for  new  mosquito  and  in  consequence 
new  human  infections.  It  is  just  as  important  a  part  of  the  preven- 
tion of  the  disease  as  any  other  single  measure. 

5.  Quinine  prophylaxis  is  an  unsatisfactory  measure  which  must 
be  used  by  travelers,  explorers  and  troops.  There  is  no  method  of 
using  quinine  which  will  entirely  prevent  malaria  when  the  chances 
for  infection  are  many;  the  following  methods  have  all  been  used:  (1) 
The  so-called  gram  prophylaxis,  in  which  one  gram  of  quinine  is  taken 
intermittently  every  tenth  day  as  the  minimum  to  every  fourth  day 
as  the  maximum.  The  gram  may  be  taken  in  a  single  dose  at  bed-time, 
or  in  four  divided  doses  during  the  day-time.  (2)  The  double  gram 
prophylaxis,  in  which  the  dose  is  taken  on  two  successive  days,  as,  for 
example,  on  the  tenth  and  eleventh,  or  on  the  fifth  and  sixth.  (3) 
The  half  gram  prophylaxis,  as  proposed  by  A.  Plehn,  was  0.5  gram- 
every  fifth  day;  experience  has  shown  that  it  is  of  little  value.  (4) 
A  daily  dose  of  0.4  to  0.8  gram  gives  better  results  than  any  other 
method,  since  the  patient  suffers  less  from  cinchonism  than  when 
larger  doses  are  taken  intermittently,  and  takes  his  quinine  more 
faithfully.  The  size  of  the  dose  depends  on  the  form  of  fever  pres- 
ent and  the  number  of  chances  for  infection;  0.4  gram  will  often 
protect  against  tertian  and  quartan,  while  even  0.8  may  fail  to  pre- 
vent aestivo-autumnal.  Latent  infections  and  relapses  among  ' '  prophy- 
lacticers"  are  common  and  black  water  fever  is  not  an  infre- 
quent sequel. 

6.  Personal  prophylaxis  by  means  of  head  nets,  gloves,  suitable 
clothing,  and  the  use  of  essential  oils,  such  as  citronella,  on  exposed 
parts  of  the  body,  is  helpful  in  emergencies. 


784  PATHOGENIC  PROTOZOA 

In  conclusion,  it  may  be  affirmed  that  any  district,  no  matter  how 
notorious,  may  be  freed  from  malaria,  if  necessity  demand  it  and 
money  be  forthcoming,  by  means  of  the  above  measures. 

Pathology. — The  pathological  features  of  the  disease  are  quite  defi- 
nite; at  autopsy  there  is  evidence  of  some  secondary  anemia,  due  to 
the  destruction  of  enormous  numbers  of  erythrocytes;  the  hemozoin, 
in  well-marked  cases,  accumulates  in  the  viscera  until  they  are  choco- 
late or  slate  colored;  the  spleen  is  enlarged  and  friable,  and  the  liver 
and  kidneys  may  show  cloudy  swelling.  In  smears  prepared  from  the 
spleen,  liver,  kidneys,  brain  and  bone  marrow,  parasites  and  hemozoin 
will  be  found,  although  the  former  may  not  be  numerous.  The  distri- 
bution of  the  parasites  is  usually  unequal,  but  they  are  often  present 
in  the  spleen  and  brain  in  greatest  numbers. 

The  origin  of  the  pigment  has  already  been  described;  it  is  phago- 
cyted  and  accumulates  in  the  viscera,  but  after  a  time  disappears  in 
some  unknown  way;  its  presence,  therefore,  is  an  indication  of  ma- 
laria in  recent  years.  It  must  be  distinguished  from  hemosiderin,  a 
yellowish  pigment  found  in  the  viscera  after  extensive  destruction  of 
red  blood  cells.  Hemozoin  is  soluble  in  alkalis  and  insoluble  in  acids, 
water,  chloroform,  alcohol  and  ether,  while  hemosiderin  is  insoluble  in 
acids  and  alkalis  but  soluble  in  alcohol.  Both  contain  iron,  yet  the 
former  (hemozoin)  does  not  give  a  Berlin  blue  reaction,  while  it  is 
present  with  the  latter. 

The  leucocytes,  although  increased  during  paroxysm,  are  soon 
diminished  so  that  leucopenia  is  characteristic  of  the  disease ;  in  addi- 
tion, there  is  a  relative  increase  in  the  mononuclear  leucocytes.  The 
loss  of  hemoglobin  is  very  great,  yet  is  quickly  recovered  from  during 
convalescence. 

In  fatal  cases  of  aestivo-autumnal  fever  a  striking  feature  is  the 
presence  of  innumerable  infected  red  cells  in  the  capillaries  of  the 
brain  or  abdominal  viscera.  A  smear  from  a  pigmented  brain  may 
show  a  capillary  thrombus  made  up  of  infected  erythrocytes,  most  of 
them  showing  the  sporulating  stage.  In  deaths  after  repeated  ma- 
larial attacks  the  kidneys  will  show,  in  addition  to  the  presence  of 
many  parasites,  a  marked  chronic  diffuse  nephritis,  one  of  the  most 
important  sequelae  of  the  disease. 

Immunity. — The  disease  is  strictly  confined  to  human  beings,  as 
none  of  the  lower  animals  are  susceptible;  there  appears  to  be  some 
racial  and  acquired  immunity,  although  it  is  incomplete;  in  native 
settlements  the  number  of  children  showing  parasites  in  the  blood  is 


SPOROZOA  785 

much  greater  than  the  number  of  adults,  yet  the  latter  are  not  abso- 
lutely immune ;  one  attack  certainly  gives  no  protection  against  a  new 
infection.  The  evidence  from  different  regions  is  quite  conflicting  in 
regard  to  immunity  and  has  been  most  plausibly  explained  in  this 
way;  where  the  disease  prevails  throughout  the  year  there  is  a  con- 
stant reinfection  and  under  this  stimulus  the  body  is  able  to  keep  up 
an  immunity  strong  enough  to  kill  off  the  parasites  before  any  symp- 
toms arise ;  in  other  regions,  however,  where  malaria  is  a  seasonal  dis- 
ease, the  constant  stimulus  is  lacking  and  there  is  less  evidence  of 
immunity.  It  has  also  been  noted  that  the  three  varieties  of  the  dis- 
ease are  distinct,  and  that  no  immunity  against  the  whole  group  is 
obtained  by  an  infection  with  one  species. 

Since  many  mild  cases  recover  without  treatment,  it  is  apparent 
that  some  little  immunity  is  produced  by  an  infection,  yet  it  is  tem- 
porary and  does  not  protect  against  repeated  relapses.  Before  the 
days  of  radical  treatment  with  quinine,  relapses  were  the  rule  and 
were  considered  an  essential  feature  of  the  disease  (Manneberg). 

Clinical  experience  teaches  that  relapses  occur  when  treatment  has 
been  insufficient,  and  especially  after  fatigue,  getting  wet  and  catch- 
ing cold,  or  after  over-heating  in  the  tropical  sun,  and  particularly 
after  a  sea  voyage  or  a  long  journey.  They  may  continue  to  occur 
in  a  region  free  from  malaria,  for  about  three  years;  in  a  malarial 
region  it  is  difficult  to  differentiate  between  relapses  and  reinfections. 
They  are  most  frequent  after  quartan,  then  tertian,  and  least  follow- 
ing aestivo-autumnal. 

The  cause  of  the  relapse  is  still  a  matter  of  discussion ;  Schaudinn 
explained  it  as  due  to  parthenogenesis,  the  macrogamete  changing  to 
a  schizont  after  expelling  a  part  of  its  nucleus  and  cytoplasm^  and  so 
starting  a  new  cycle  of  asexual  parasites.  Craig  has  suggested  an 
intra-corpuscular  conjugation  as  the  beginning  of  a  new  crop  of  para- 
sites. It  is  possible  that  the  old  theory,  that  parasites  survive  for 
long  periods  in  the  viscera,  is  correct  and  that  the  relapse  is  brought 
about  by  any  condition  which  temporarily  reduces  the  convalescent's 
immunity,  such  as  fatigue,  etc.  H.  C.  Clark,  in  Panama,  has  exam- 
ined a  large  series  of  placentas  from  apparently  healthy  women  and 
found  that  not  infrequently  they  contained  plasmodia  in  large  num- 
bers, even  in  the  absence  of  recent  malarial  attacks.  There  was,  evi- 
dently, sufficient  immunity  to  hold  the  parasites  in  check  under  ordi- 
nary circumstances. 


786  PATHOGENIC  PROTOZOA 

BLACK  WATER  FEVER 

This  condition  follows  malaria  and  occurs  only  in  malarial  dis- 
tricts, and  by  most  authorities  is  believed  to  be  a  sequel  of  the  disease, 
due  to  some  unknown  factor.  Castellani  and  Chalmers,  however, 
separate  it  into  three  forms,  symptomatic,  toxic  and  specific  hemo- 
globinuria, examples  of  which  are,  respectively,  malarial  and  quinine, 
hemoglobinuria  and  black  water  fever.  In  none  of  these,  however,  is 
the  etiology  clear;  the  disease  does  not  occur  independently  of  ma- 
laria, and  an  attack  may  be  precipitated  or  aggravated  by  quinine. 
Leishman  has  described  certain  cell  inclusions,  possibly  chlamydozoa, 
as  the  cause.     The  etiology  is,  at  the  present  time,  far  from  clear. 

TREATMENT  OF  MALARIA 

In  quinine  we  have  a  true  chemical  specific  for  malaria,  and  when 
given  early  enough  and  in  sufficient  doses,  will  cure  the  disease  with 
certainty.  It  is  usually  given  in  the  form  of  the  sulphate  or  dihydro- 
chlorate,  preferably  in  solution,  but  may  be  administered  in  freshly 
prepared  capsules;  pills  and  tablets,  while  convenient,  are  unsatis- 
factory because  of  their  relative  insolubility.  It  acts  vigorously  on 
the  merozoites  and  young  trophozoites,  but  has  almost  no  direct  effect 
upon  the  gametes.  The  size  of  the  dose  depends  on  the  form  of  the 
fever  and  its  severity.  In  ordinary  cases  five  grains  three  times  a  day 
is  sufficient,  while  in  severe  infections  not  less  than  thirty  grains  a  day 
must  be  given;  the  best  time  is  immediately  after  meals,  without  re- 
gard to  the  time  of  the  chill.  To  prevent  relapses,  the  treatment  of  the 
original  infection  must  be  thorough,  and  the  patient  should  be  kept 
in  bed,  upon  a  light  diet  and  attention  paid  to  the  condition  of  the 
bowels.  During  and  after  convalescence  the  treatment  must  be  con- 
tinued for  three  months,  though  the  daily  dose  may  be  decreased 
gradually,  beginning  a  week  or  ten  days  after  the  subsidence  of  the 
fever.  In  exceptional  cases  a  relapse  will  occur  while  the  patient  is 
still  taking  massive  doses  of  quinine,  and  by  some  this  has  been  looked 
upon  as  an  evidence  of  immunity  of  the  parasite  to  the  drug,  but  it  is 
possible  that  it  is  merely  due  to  non-absorption  of  the  quinine,  and 
carminatives  should  be  added  to  the  dose  to  assist  in  its  absorption. 

OTHER  MALARIAL  PARASITES 

From  time  to  time  additional  malarial  parasites  have  been  de- 
scribed, only  two  of  which  are  of  any  importance  at  present:    Plas- 


SPOROZOA 


787 


modium  vivax,  variety  minuta,  and  Plasmodium  tenue.  The  former 
was  described  by  Ahmed  Emin  in  1914.  It  resembles  the  usual  ter- 
tian in  general,  but  differs  in  the  following  points;  it  is  smaller,  the 
infected  erythrocyte  is  not  enlarged,  and  the  number  of  merozoites  is 
small  (four  to  ten).  The  pigment  is  fine  and  motility  is  not  marked. 
Multiple  infection  of  the  erythrocytes  is  not  uncommon.  Craig  ^  de- 
scribed a  similar  parasite  in  1900,  and  he  suggests  that  the  parasite 
has  been  confounded  with  Plasmodium  malarise. 

In  1914,  Stephens  ^  described  an  organism  from  one  slide,  which  he 
calls  Plasmodium  tenue.  It  is  said  to  be  deficient  in  pigment,  mark- 
edly motile,  and  rich  in  chromatin.  Since  the  parasite  Vas  described 
from  one  slide,  it  is  very  doubtful  if  it  can  at  present  be  accepted  as 
a  valid  species. 

Piroplasmidse  (Franja). — This  is  a  provisional  family  belonging  to 
the  hemosporidia,  the  type  of  which  is  Babesia  higeminum,  Smith 
and  Kilborne   {pirosoma,  pi- 
roplasma) ,    the    cause    of 
Southern  cattle  fever. 

The  parasite  was  first  de- 
scribed by  Smith  and  Kil- 
borne in  1889,  and  correctly 
placed  by  them  among  the 
protozoa;  they  also  demon- 
strated its  transmission  by 
the  cattle  tick,  and  this 
achievement  marks  the  be- 
ginning of  medical  protozo- 
ology. To  the  original  para- 
site, the  cause  of  Texas  fever, 
has,  in  course  of  time,  been 
added  other  forms  until  now 
we  have  a  family  consisting 
of  Babesia  higeminum,  hovis, 

canis,  equi,  ovis,  mutans,  quadrigeminum,  and  a  closely  related  para- 
site, Theileria  parva. 

Morphology. — The  parasites  are  pear-shaped,  round,  oval  or  ame- 
boid, inhabiting  mammalian  red  blood  cells,  which  they  destroy  but 


Fig.  196. — Babesia  bigeminum.  (Army  Med. 
School  Collection,   Washington,   D.  C.) 


»  Craig,  Jour.  Parasitol.,  Urbana,  111.,  1914,  1,  88. 

*  Stephens,  Proc.  Roy.  Soc,  Lond.,  Series  B,  87.  p.  375. 


788  PATHOGENIC  PROTOZOA 

without  producing  pigment.  In  cattle,  sheep,  horses  and  dogs,  the 
freed  hemoglobin  is  excreted  by  the  kidneys,  producing  the  disease 
variously  known  as  red  water  fever,  Texas  or  Southern  cattle  fever, 
tick  fever,  bovine  malaria,  hemoglobinuria,  and  others.  The  parasite 
is  small,  two  to  four  microns  long  and  one  to  two  wide,  and  character- 
istically occurs  in  pairs,  the  narrow  ends  being  united.  When  the 
parasite  is  mature  the  two  daughter  cells  separate  and  when  liberated 
by  the  degenerated  erythrocyte  attack  new  red  cells.  The  pear-shaped 
babesia  enters  a  new  cell  by  its  broad  end,  becomes  rounded  or  ring- 
like, then  ameboid  in  form,  and  finally  the  nucleus  sends  out  a  bud ; 
this  divides  irrto  two  by  forking,  and  as  the  nuclear  matter  continues 
to  grow  each  portion  becomes  surrounded  by  cytoplasm  and  ultimately 
the  two  new  daughter  cells  separate  from  one  another.  Multiple  in- 
fections of  single  red  cells  are  common,  as  many  as  sixteen  pairs 
having  been  seen  in  a  single  cell. 

Good  preparations  for  clinical  work  are  obtained  by  some  one  of 
the  Romanowski  stains,  but  the  finer  details  of  the  nucleus  and  cell 
division  can  only  be  studied  after  iron  hemotoxylin  staining. 

The  parasites,  with  the  exception  of  Theileria  parva,  do  not  disap- 
pear completely  from  the  blood  after  the  animal  recovers  from  its 
illness,  but  remain  indefinitely  in  the  circulation,  and  their  continued 
presence  and  virulence  may  be  demonstrated  by  the  inoculation  of 
non-immune  animals  with  blood  from  an  animal  which  has  recovered. 

Transmission. — Transmission  from  host  to  host  is  by  means  of  ticks. 
Margaropus  annulatus  carries  Southern  cattle  fever  in  the  United 
States,  but  other  ticks  are  carriers  in  South  America  and  the  West 
Indies.  In  general,  each  species  of  hahesia  is  specific  for  a  particular 
animal,  and  each  is  carried  by  a  separate  group  of  ticks. 

Clinical  Ohservaiions. — Clinical  observations  have  shown  an  in- 
cubation period  of  about  fourteen  days;  the  onset  is  with  fever  and 
the  cattle  look  weak  and  ill,  neither  eat  nor  chew  the  cud,  but  stand 
with  sunken  head  and  relaxed  ears.  A  bloody  diarrhea  sets  in 
early  and  the  urine,  which  is  small  in  amount,  is  deep  red  in  color 
and  contains  much  albumin.  The  blood  shows  few  parasites  at  first, 
but  they  soon  increase  and  the  number  of  red  cells  falls  rapidly.  The 
mortality  varies  in  different  epidemics  from  five  to  sixty  per  cent, 
but  has  been  as  high  as  ninety  in  some  herds.  Young  cattle,  under  a 
year,  have  a  mild  form  of  the  disease,  and  remain  thereafter  immune. 

The  treatment,  once  the  disease  has  appeared,  is  unsatisfactory, 
since  we  have  no  specific,  but  much  may  be  done  by  prophylaxis. 


SPOROZOA  789 

Quarantine  of  cattle  from  infected  regions  is  absolutely  necessary  to 
prevent  the  spread  of  the  disease,  since  all  immune  animals  are  also 
carriers.  Young  animals,  between  nine  and  twelve  months  of  age, 
may  be  inoculated  with  the  blood  of  those  which  have  recovered,  five 
to  ten  c.c.  being  given  in  a  single  dose;  the  disease  lasts  two  to  three 
days,  and  the  mortality  is  not  great,  as  eighty  to  ninety  per  cent  of  the 
inoculated  animals  recover  and  remain  immune.  Inoculation,  how- 
ever, is  a  measure  of  doubtful  value  and  some  means  of  tick  eradi- 
cation should  be  used  if  the  disease  is  to  be  permanently  stamped 
out. 

lAfe  History  of  the  Tick  {Mar gar  opus  annulatusY 

The  tick 's  life  is  divided  into  two  stages,  one  part  passed  on  cattle 
and  another  part  passed  on  the  ground.  The  mature  female,  as 
found  on  cattle,  is  about  half  an  inch  in  length,  plump  and  olive  green. 
When  fully  engorged  with  blood  from  its  host,  it  drops  to  the  ground, 
seeks  a  sequestered  hiding  place  and  if  it  escapes  birds,  ants  and 


Fig.  197. — The  Texas  Fever  Tick  (Margaropus  annulatus).    (Rosenau,  "Preven- 
tive Medicine  and  Hygiene.") 

other  enemies,  begins  after  a  few  days  of  warm  weather  to  lay  eggs. 
These  are  small,  elliptical,  at  first  light,  later  dark  brown,  and  are 
cemented  together  in  irregular  masses  by  a  sticky  secretion ;  in  num- 
bers they  vary  from  a  few  hundred  to  more  than  .five  thousand  for 
each  female.  The  female  tick  dies  in  a  few  days  after  the  egg-laying 
has  been  completed.  The  eggs  soon  hatch  (after  nineteen  days  in 
sujnmer  to  one  hundred  and  eighty-eight  in  winter)  and  a  small,  oval, 

1  Farmers'  BuU.  No.  498,  U.  S.  Dept.  of  Agri. 


790  PATHOGENIC  PROTOZOA 

six-legged  larva  or  seed  tick  appears,  and  promptly  climbs  up  on  the 
nearest  vegetation,  grass,  weeds  or  bushes  to  lie  in  wait  for  a  warm- 
blooded host.  Although  while  on  vegetation  seed  ticks  do  not  take 
food  nor  grow,  their  endurance  is  great,  and  during  the  colder  parts 
of  the  year  they  may  live  for  eight  months.  The  next  stage  begins 
after  the  seed  tick  has  found  a  host,  when  it  sucks  blood,  increasing 
in  size  and  soon  (five  to  twelve  days)  molts  and  a  new  form,  the  eight- 
legged  nymph,  appears.  In  five  to  eleven  days  more  a  second  molt 
occurs  and  the  tick  is  then  sexually  mature  and  males  can  be  dis- 
tinguished from  females.  The  female  does  not  move  about  on  the 
animal,  but  the  male  seeks  her  out,  and  after  fertilization  growth  goes 
on  rapidly  until  the  engorged  female  drops  to  the  ground.  To  sum- 
marize :  On  the  ground  is  the  engorged  female,  the  eggs  and  seed  ticks ; 
on  the  animal  is  the  seed  tick,  the  nymph,  the  sexually  mature  adult 
and  finally  the  engorged  female.  The  infected  female  tick  transmits 
the  babesia  to  the  larvjB  through  the  eggs,  but  does  not  herself  bite 
nor  convey  the  disease  directly  to  another  animal. 

It  is  evident  that  the  tick  may  be  attacked  in  the  pasture  or  on 
the  cattle.  Pasture  rotation  is  one  of  the  methods  recommended  by 
the  Agricultural  Department,  and  it  rests  upon  the  fact  that  all  the 
ticks  will  die  from  starvation  in  an  unused  pasture  in  from  six  to 
twelve  months,  varying  with  the  climate,  the  shorter  period  holding 
true  for  warmer  localities.  By  changing  pastures  a  farm  may  be  freed 
of  ticks  in  four  and  a  half  or  eight  months,  depending  on  the  plan  fol- 
lowed. For  economic  reasons,  lack  of  sufficient  pasture  land,  etc.,  this 
plan  has  not  been  widely  adopted.  A  second  plan,  that  of  dipping, 
has  been  more  successful.  The  cattle  are  driven  through  a  large  dip- 
ping vat  at  intervals  of  two  weeks  (never  more  than  three  must 
elapse)  until  they  and  the  pasture  are  free  from  ticks.  The  fluid  in 
.the  dipping  vats  is  an  alkaline  solution  of  arsenic ;  oil  dips  are  little 
used  at  present.  Arsenical  dips  are  cheap,  easily  prepared  and  effi- 
cacious, two  or  at  most  three  dippings,  at  ten-day  intervals,  being  suf- 
ficient to  free  heavily  infected  cattle,  and  if  they  can  then  be  put  on 
tick-free  pastures  the  problem  is  solved.  If  no  tick-free  pastures  are 
available,  dipping  must  be  continued,  as  above,  until  the  animals 
remain  permanently  free.. 

The  prevention  of  tick-borne  disease  is  to  be  solved,  therefore,  by 
tick  eradication,  and  experience  in  the  Southern  states  has  shown  this 
to  be  a  practical  measure. 


SPOROZOA  791 

SUB-CLASS— NEOSPORIDA 
SARCOSPORIDIA 

These  organisms  belong  to  the  sub-class  Neosporidia  of  the  Sporo- 
zoa,  because  the  spore  formation  commences  before  the  completion 
of  growth.  They  have  been  known  since  1843,  when  Miescher  dis- 
covered ** tubes''  in  muscle  fibers,  visible  to  the  naked  eye  as  fine, 
white,  opaque  filaments.  They  have  been  found  in  deer,  cattle,  sheep, 
swine,  rabbits  and  man,  and  occasionally  in  birds  and  reptiles.  Al- 
though long  known,  our  knowledge  of  them  is  still  defective.  In  sheep 
they  are  the  cause  of  severe  epizootics,  and  in  mice  they  are  fatal: 
otherwise  they  seem  to  be  harmless  parasites  and  in  rare  instances 
have  been  accidentally  found  at  autopsy  in  man. 

The  method  of  transmission  is  not  definitely  known.  Theobald 
Smith  was  able  to  infect  mice  with  Sarcocystis  muris  by  feeding  mus- 
cles from  infected  mice,  and  Darling  was  able  to  infect  guinea-pigs 
in  the  same  manner.  In  nature  it  is  probable  that  infection  occurs 
through  the  intestinal  tract.  They  are  exceedingly  common  in  some 
localities  and  may  be  seen  in  most  abattoirs ;  they  have  been  found  in 
ninety-eight  per  cent  of  swine,  ninety-eight  per  cent  of  sheep,  and 
commonly  in  deer  and  mice. 

To  the  naked  eye  they  appear  as  whitish,  opaque,  cylindrical  bod- 
ies in  the  muscles  lying  parallel  to  the  fibres.  In  the  sheep,  Sarcocystis 
tenella  reaches  a  length  of  sixteen  mm.,  and  in  the  deer  cysts  of  fifty 
mm.  are  found.  In  structure  the  *  *  tube ' '  is  seen  under  the  microscope 
to  be  composed  of  many  sickle-shaped  spores,  called  Rainey's  cap- 
sules. The  tube  has  not  a  single  cavity  but  a  honey-comb  or  alveolar 
structure,  and  the  spores  are  found  in  small  aggregations  in  the  cham- 
bers, completely  walled  off  from  one  another.  The  tube  itself  has  a 
heavy  striated  wall,  either  secreted  by  the  organism  or  composed  of 
altered  muscle  fiber  of  the  host.  The  spores  are  sickle  or  kidney- 
shaped  and  vary  both  among  themselves  and  with. the  host.  They 
contain  a  nucleated  trophozoit,  and  at  one  pole  either  a  clear  or  an 
obliquely  striated  body. 

Laveran  and  Mesnil  (1899)  obtained  a  toxin  from  Sarcocystis 
tenella  and  named  it  sarcocystin.  Darling,  working  in  Panama,  found 
this  organism  in  the  biceps  muscle  of  a  negro  from  Barbados. 


CHAPTER   LVIII 

CLASS  IV— INFUSORIA 
SUB-CLASS— (CILIATA) 

This  class  is  sharply  distinguished  from  all  other  protozoa  by  the 
presence  of  numerous  cilia,  distributed  in  various  ways  over  the 
ectoplasm,  which  serve  as  organs  of  locomotion,  and  by  the  presence 
of  two  or  more  nuclei.  They  fall  into  two  classes,  the  ciliata,  which 
are  provided  with  cilia  during  the  entire  life  cycle,  and  the  suctoria, 
which  lose  their  cilia  on  entering  the  adult  stage;  the  latter  are  of 
no  interest,  since  none  are  holozoic. 

Only  one  of  the  ciliates  is  of  importance  in  medicine,  Balantidium 
coll.  Balantidium  and  Nyctotherus  faba,  having  been  reported  only 
once  in  man  by  Schaudinn,  are  too  rare  to  be  considered  here. 

BALANTIDIUM  COLI  (Malstan) 

This  parasite,  first  described  in  1857,  is  commonly  found  in  the 
lower  intestinal  tract  of  swine;  while  usually  harmless,  it  may  cause 
a  moderate  mortality  in  .them  from  subacute  and  chronic  dysentery. 
It  has  frequently  been  reported  as  present  in  man,  and  is  readily 
diagnosed  on  examination  of  the  stools. 

The  organism  is  much  like  Paramecium  and  is  actively  motile  when 
obtained  from  fresh  stools,  or  from  scrapings  from  the  ulcerated 
cecum  at  autopsy.  The  body  is  ovoid  and  rather  stumpy  toward  the 
anterior  end,  distinguished  by  the  triangular  peristome;  average 
measurements  are  about  eighty  by  sixty  microns.  The  ectoplasm  is 
covered  with  thick,  parallel  bands  of  active  cilia,  giving  the  animal 
a  striated  appearance;  the  macronucleus  is  kidney  shaped  and  lying 
close  to  it;  the  micronucleus  may  usually  be  distinguished.  At  one 
side  of  the  organism  are  found  two  contractile  vacuoles,  and,  in  the 
endoplasm,  food  vacuoles  and  fat  droplets. 

Multiplication  in  its  simplest  form  consists  of  binary  division, 
though  conjugation  also  occurs.     In  the  feces  of  the  host  encysted 

792 


INFUSORIA 


793 


forms  are  common,  and  by  means  of  these  the  infection  is  transferred 
to  new  hosts. 

Although  long  believed  to  be  harmless,  pathological  lesions  have 
been  found  by  Strong  and  others  in  man  and  monkeys,  consisting  of 


Fig.  198. — Balantidium  coli  in  a  Follicle  of  the  Colon,  Breaking  Through 
THE  Mucosa.    (Army  Med.  School  CoUection,  Washington,  D.  C.) 

thickening  and  ulceration  of  the  infected  intestine,  with  penetration 
of  the  balantidium  through  the  gut  wall  to  the  subserous  layer.  Exten- 
sive lesions  may  be  found  at  autopsy,  even  in  the  absence  of  a  his- 
tory of  dysentery  or  diarrhea. 


CHLAMYDOZOA 

There  is  a  small  group  of  very  minute  intracellular  organisms 
which  are  believed  by  many  to  be  protozoa,  and  for  them  von  Pro- 
wazek  has  created  the  class  named  '  *  chlamydozoa. ' '  He  believes  that 
such  organisms  produce  smallpox,  vaccinia,  hydrophobia,  trachoma, 
scarlatina,  Molluscum  contagiosum,  avian  plague,  the  contagious  epi- 


794  PATHOGENIC  PROTOZOA 

thelioma  of  birds,  hoof  and  mouth  disease,  jaundice  of  silk  worms 
and  others.  The  delicate  organisms  are  first  noticed  as  fine  dots  of 
chromatin  lying  in  the  cytoplasm  of  the  infected  cell  (the  so-called 
elementary  bodies).  When  larger  they  are  called  initial  bodies  and 
are  believed  to  set  up  a  reaction  in  the  cell,  which  causes  an  extru- 
sion of  the  plastin  from  the  nucleus;  this  envelops  the  initial  bodies 
like  a  mantle,  hence  the  name  ^'chlamydozoa,^'  from  the  Greek  stem 
''chlamys,"  meaning  mantle.  Inside  of  this  covering  they  multiply, 
in  some  instances  invading  the  nucleus. 

Their  very  existence  is  a  matter  of  dispute,  yet  at  the  present  time 
the  consensus  of  belief  is  that  they  constitute  a  group  of  parasites 
and  are  not  merely  forms  of  cell  inclusions  or  degenerations. 

Nogouchi  ^  has  shown  that  in  trachoma  these  organisms  may  be 
found  alone  without  any  other  pathogenic  organisms  being  present, 
and  that  they  may  be  transferred  to  the  conjunctiva  of  the  baboon 
and  higher  apes,  producing^  a  mild  form  of  the  disease.  Smears  of 
the  exudate,  taken  from  the  infected  animals  and  stained  with  Giemsa, 
show  cell  inclusions  without  the  presence  of  bacteria. 

1  Nogouchi,  Jour.  Exp.  Med.,  N.  Y.,  1915,  xxii,  304. 


CHAPTER   LIX 
TECHNIQUE  OF  BLOOD  EXAMINATIONS  FOR  PROTOZOA 

Slides  and  cover  glasses  must  be  scrupulously  clean,  and  are  best 
kept  in  covered  glass  jars  which  are  dust-proof.  For  most  purposes 
stained  preparations  on  slides  are  satisfactory  and  much  easier  to 
handle  than  cover  glasses. 

In  all  investigations,  both  wet  and  dry  preparations  should  be 
made ;  some  practice  is,  of  course,  necessary  to  obtain  satisfactory  ones, 
thin  enough  to  show  details.  As  Manson  says,  in  looking  for  dates 
on  coins,  one  would  not  pile  one  coin  on  top  of  another;  so,  in  search- 
ing blood  cells  for  parasites,  it  is  necessary  to  have  the  cells  lying 
flat,  and  in  a  single  layer.  Wet  preparations  are  made  with  a  drop  of 
blood  not  much  larger  than  the  head  of  a  pin,  on  which  a  cover  glass 
is  dropped,  and  gently  pressed  down;  it  is  advantageous  to  lute  the 
margin  of  the  cover  glass  with  warm  vaseline  (warmed  by  holding 
the  cameFs  hair  pencil  over  a  flame  for  a  few  seconds)  to  prevent 
evaporation  and  consequent  currents  under  the  cover  glass.  A  prop- 
erly sealed  wet  preparation  may  be  examined  at  intervals  during  sev- 
eral days,  and  this  is  often  necessary  in  studying  movements  and  the 
life  cycle.  Malarial  parasites,  trypanosomes  and  others  are  quickly 
detected  by  their  movements. 

Stained  preparations  are  necessary  for  the  study  of  details  of 
structure,  and  for  clinical  work  are  invaluable,  since  smears  may  be 
collected  at  the  bedside,  and  examined  later  at  home,  either  by  day- 
light or  artificial  light.  In  general,  a  one  hundred  watt  concentrated- 
filament  nitrogen  bulb  will  be  found  the  best  source  of  illumination 
for  the  study  of  both  living  and  stained  protozoa.  The  Welsbach 
mantle,  acetylene  gas  or  even  a  kerosene  lamp  are  satisfactory  sub- 
stitutes. At  times  a  color  screen,  made  by  interposing  a  round,  glass 
flask,  filled  with  dilute  copper  sulphate  solution,  between  the  light  and 
the  mirror,  will  prove  advantageous.  It  is  particularly  in  the  South 
and  in  the  tropics  that  artificial  illumination  has  been  most  valuable. 

To  prepare  stained  preparations,  a  small  drop  of  blood  from  the 

796 


796  PATHOGENIC  PROTOZOA 

ear  is  placed  on  a  slide  near  one  end,  while  with  another  clean  slide 
the  spread  is  made  by  holding  its  narrow  end  in  the  drop  until  the 
blood  has  flowed  between  the  two,  and  then  pushing  the  spreader,  or 
second  slide,  held  at  an  angle  of  about  45°,  toward  the  opposite  end 
of  the  first  slide.  The  drop  of  blood  follows  closely  behind  the 
spreader,  and  the  thickness  of  the  film  may  be  varied  by  changing  the 
angle  of  the  spreader.  The  thin  film  of  blood  dries  quickly  in  the 
air.  It  is  never  fixed  in  the  flame,  but  always  in  methyl  alcohol  or  in 
the  stain  itself. 

Many  varieties  of  blood  stains  have  been  proposed,  but  the  simplest 
and  best  are  those  of  Wright  and  MacNeal.  Wright's  stain  is  pre- 
pared as  follows: 

Dissolve  0.5  gm.  of  sodium  bicarbonate  in  100  c.c.  distilled  water, 
and  add  1  gm.  of  methylene  blue  (Gruebler).  Any  of  the  methylene 
blues  of  Gruebler  known  as  ''BX,"  Koch's  or  Ehrlich's  rectified  may 
be  used.  It  seems  to  be  important  that  the  bicarbonate  of  soda  be  all 
dissolved  before  adding  the  methylene  blue. 

The  mixture  is  next  to  be  steamed  in  an  ordinary  steam  sterilizer 
for  one  hour,  counting  the  time  after  "the  steam  is  up."  The  heating 
should  not  be  done  in  a  pressure  sterilizer,  or  in  a  water-bath,  or  in 
any  other  way  than  as  stated.  This  steaming  of  the  alkaline  solution 
of  methylene  blue  effects  certain  changes  in  the  methylene  blue  where- 
by a  polychromatic  quality  is  given  to  it,  so  that  the  compound  with 
eosin,  which  is  later  to  be  formed  with  it,  has  tlie  property  not  only 
of  differentially  staining  the  chromatin  of  the  malarial  parasite,  but 
also  of  differentiating  and  bringing  out  more  sharply  the  nuclei  and 
granules  of  the  white  blood  corpuscles. 

When  the  steaming  is  completed,  the  mixture  is  removed  from  the 
sterilizer  and  allowed  to  cool,  the  flask  being  placed  in  cold  water  if 
desired.  When  it  is  cold,  without  filtering,  pour  it  into  a  large  dish 
or  flask,  and  add  to  it,  stirring  or  shaking  meanwhile,  a  sufficient 
quantity  of  a  1:1000  solution  of  eosin  (Gruebler,  yellowish,  soluble 
in  water)  until  the  mixture,  losing  its  blue  color,  becomes  purplish, 
and  a  scum  with  yellowish  metallic  luster  forms  on  the  surface,  while 
on  close  inspection  a  finely  granular  black  precipitate  appears  in 
suspension.  This  will  require  about  5Q0  c.c.  of  the  eosin  solution  for 
100  c.c.  of  alkaline  methylene  blue  solution.  (The  proper  amount 
of  eosin  to  add  may  be  determined  by  placing  a  drop  of  mixture  on 
white  filter  paper,  and  adding  eosin  until  the  blue  spot  shows  a  dis- 
tinct halo  of  pink,) 


BLOOD  EXAMINATION^  FOR  PROTOZOA  797 

The  precipitate  is  collected  on  a  filter,  and,  Avithout  washing,  is 
allowed  to  dry  thereon ;  when  thoroughly  dry,  dissolve  this  precipitate 
in  pure  methyl  alcohol  in  the  proportion  of  three-tenths  of  a  gram  to 
one  hundred  cubic  centimeters  of  alcohol.  This  alcoholic  solutoin  is 
the  staining  fluid. 

MacNeal  ^  has  shown  that  both  methylene  azure  and  methylene 
violet  participate  in  the  nuclear  staining,  and  that  an  excellent  stain, 
equal  to  any,  may  be  made  directly  by  mixing  the  pure  dyes  accord- 
ing to  the  following  formula : 

Solution  A.     Methylene  azure 0.3  gm. 

Methylene   violet    (Bernthsen's,   insoluble   in 

water)    0.1 

Methylene  blue    2.4 

Methyl  alcohol,  pure  (Merck's  reagent) 500.0 

Solution  B.     Eosin,  yellowish,  water  soluble 2.5 

Methyl  alcohol,  pure 500.0 

These  stock  solutions  will  keep  for  at  least  a  year.  They  are  mixed 
as  needed  in  equal  parts,  and  diluted  by  the  addition  of  25  c.c.  of 
methyl  alcohol  to  each  100  c.c.  of  the  mixture.  The  mixture  will 
keep  for  several  months. 

The  method  of  staining  is  the  same,  whatever  methyl  alcohol  stain 
be  used.  The  cover  glass  or  slide  is  flooded  with  the  stain,  which  is 
allowed  to  act  for  one  minute ;  next,  as  much  distilled  water  is  added, 
drop  by  drop,  as  the  slide  will  hold,  or  until  a  yellowish  metallic 
scum  is  formed  on  the  surface,  and  the  mixture  is  allowed  to  act 
from  three  to  five  minutes.  A  convenient  staining  dish  is  made  by 
laying  a  pair  of  glass  rods  on  top  of  a  flat  oblong  dish ;  the  rods  may 
be  held  in  place  with  adhesive  plaster  or  perforated  strips  of  wood. 
Fixation  is  accomplished  by  the  undiluted  solution,  but  the  actual 
staining  does  not  occur  until  water  is  added ;  by  repeated  washing  in 
distilled  water,  any  desired  differentiation  may  be  made. 

It  is  sometimes  convenient,  when  searching  for  malarial  parasites 
in  clinical  cases,  to  stain  as  follows :  ^ 

Use  four  bottles  or  Coplin  jars;  in  the  first,  put  the  undiluted 
stain,  and  in  it  immerse  the  slide  for  one  minute ;  in  the  second,  put 
distilled  water  and  transfer  the  slide  to  it  for  four  or  five  minutes; 

1  MacNeal,  Jour.  Inf.  Dis.,  Chicago,  1906,  iii,  412. 

2  Russell,  F.  F.y  Jour.  Amer.  Med.  Ass.,  Chicago,  1915,  Ixiv,  2131. 


798  PATHOGENIC  PROTOZOA 

in  the  third  jar  put  diluted  Hanson's  stain,  about  0.5  c.c.  to  50  c.c. 
water,  and  in  this  the  smear  remains  one-half  minute;  in  the  fourth 
bottle  or  tumbler  put  distilled  water,  in  which  the  slide  is  washed 
quickly  and  then  dried.  This  method,  for  the  average  man,  is  more 
economical  and  simpler  than  the  open  method.  Stained  smears  are 
examined  directly  in  immersion  oil  without  the  use  of  cover  glass. 

Manson's  stain  is  prepared  as  follows: 

Two  grams  of  methylene  blue,  medicinally  pure  (Hochst),  is 
added  to  100  c.c.  of  a  boiling  5  per  cent  solution  of  borax.  This  stain, 
though  not  permanent,  will  last  a  long  time.  It  is  used  chiefly  for 
the  diagnosis  of  malaria.  It  is  diluted  before  use.  Stain  for  ten  to 
fifteen  seconds. 

When  parasites  are  few,  as  in  latent  malaria,  thick  films  may  be 
used,  as  first  proposed  by  Ross.  A  large  drop  of  blood  is  spread 
thickly  on  the  side,  with  the  needle  or  pen  used  in  puncturing  the 
ear ;  after  drying,  it  is  put  into  (1)  95  per  cent  methyl  alcohol  to  which 
1  per  cent  of  hydrochloric  acid  has  been  added,  until  the  smear  has 
been  hemolized;  (2)  it  is  then  thoroughly  washed  in  running  water, 
(3)  and  then  stained  in  the  usual  way  with  a  methyl  alcohol  stain. 
Although  the  method  takes  considerable  practice,  it  is  a  valuable  pro- 
cedure. 


INDEX  OF  AUTHORS 


Abbott,  97,  326 

Abel,   189,  450,  452,  557 

Abel  and  Claussen,  585 

ACHABD     AND     BeNSAUDE,     430 

Adami,  392 

Adami  and  Chapin,  695 

Adie,  Helen,  761 

Adleb,  302 

Agbomonte,  669,  670 

Albbecht   and   Ghon,    376,   377,    378, 

555,  561 
Allegbi,  502 
Alvabez,   720 
Alvabez  and  Tavel,  503 
Andebson,  J.  G.,  707 
Andebson  and  Goldbebgeb,  677 
Andebson  and  McClintic,  80 
Andbews  and  Hobdeb,  349 
Abistotle,  2 
Abloing,  63 

Abloing  and  Chauveaux,  471 
Abloing,  Cobnevin  and  Thomas,  468 
Abloing,  Leclainche,  and  Vall^e,  468 
Abnheim,  679 
Abning,  509 

Abonson,  91,  346,  347,  349 
Abbhenius  and  Madsen,  211 
Abthus,  297 
Abustamoff,  619 
Asakawa,  214 

ASCOLI  AND   FiGABI,  201 
ASHFOBD,    641 
AVEBY,  364 

Axenfeld,  546 

Babes,  11,  510,  514,  550,  613,  614,  623, 

648 
Babes  and  Lepp,  197 
Baehb,  678 
Baginsky,  343,  460,  518 


Baginsky  and  Sommebfeld,  343,  344, 

676 
Bail,  292,  330 
Bail  and  Pettebson,  292 
Bail  and  Weil,  292 
Baldwin,  494 

Bandelieb  and  Roepke,  497 
Bandi  and  Simonelli,  596 
Bang,  707 
Banzhaf,  219,  220 
Babkeb  and  Cole,  424 
Basenau,  430,  705 
Bauee,    269 

Baumgabten,  106,  479,  486 
Baumleb,  540 

Beck,  493,  499,  514  , 

Beck,  M.,  Kolle  and  Wasseemann,517 
Beckmann,  78 
Behbens,  719 
V.   Behbing,  76,   77,  78,  79,   195,  205, 

221,  295,  487,  490,  569 
V.  Behbing  and  Kitasato,  196,  198 
V.  Behbing  and  Kitashina,  295 
V.  Behbing  and  Webnicke,  196,  198 
Beijaeff,   198 

Beijebinck,  26,  27,  703,  715 
Beinstock,  393,  481 
Belfanti,  458 

Belfanti  and  Caebone,  200 
Bebaneck,  493 
Bebestnew,  618,  620,  626 
Bebtabelli,  501,  590,  602,  603 
Beethelot,  54 

Bebtband  and  Weisweillee,  717 
Besbedka,  298,  301,  345,  416 
Beseedka  and  Steinhabdt,  298 
Beueemann  and  Gougebot,  645 
Bezancon,  334 

BEZANgoN,  Gbiffon,  and  Le  Soubd,  548 
Biggs  and  Pabk,  231 


799 


800 


INDEX  OF  AUTHORS 


Billroth,  7 

BiNAGHI,   368 
BiRCH-HlRSCHFELD,    192,   392 

Bitter,  31,  168,  503 
Blaise  and  Sambac,  64 
Blumer,  411 

BOGART  AND  BeRNHARD,   201 
BOLAND,   579 

Bollinger,  622 

Bolton,  24 

BoRDET,   200,   224,   225,  228,   232,   236, 

240,  243,  244,  337,  346,  368,  545 
BoRDET  AND  Gengou,  245,  262,  543 

BoRDET   AND  MaY,  262 

Bordoni-Uffreduzzi,  358 
BoRissow,  328 

EORSIEKOW,    139 
BOSCHETTI,   707 
BOSTROEM,    624 
BOYCE  AND    WOODHEAD,    711 

Beau  and  Denier,  589 

Brauell,  6 

Brieger,  185,  195,  415 

Brieger  and  Boer,  462,  522 

Brieger  and  Cohn,  461,  462 

Brieger  and  Frankel,  521 

Brieger  and  Kempner,  478 

Brill  and  Libman,  579 

Brougiiton,  427 

Browning,  Zinsser  and  Johnson,  243 

Bruce,  550,  755 

Bruck,  215 

Brudzinski,  716 

V.  Brunn,  90 

Buchner,   22,   63,    152,   186,   198,   203, 

224,   225,   389,  691 
Buchner  and  Hahn,  492 
Buchner  and  Meisenheimer,  50 
BuDD,  399 
Budinger,  327 
Buerger,  99,  349,  350,  355 

BUFFON,   4 

Bullock  and  Atkin,  282,  284 
Bullock  and  Hunter,  581 
Bullock  and  Western,  282 
BuMM,  380,  381 

BUNGE  AND  TrAUTENROTH,   106 

Burkholtz,  63 


BussE,  631 

Butschli,  11,  12 

Buxton,  428,  429 

Buxton  and  Coleman,  180,  431 

Caqniard-Latour,  3 

Calkins,  650,  658 

Calmette,  199,  203,  494 

Calmette  and  Guerin,  660,  662 

Canalis,  189 

Canfora,  460 

Cantacuz^ne,  280 

Cantani,  539 

Capps  and  Miller,  706 

Carlo  and  Rattone,  456 

Carriere,  47 

Carroll,  671 

Carter,  627 

Castellani,  234,  405,  614 

Castle,  415 

Certes,  34 

Chamberland  and  Roux,  194,  569,  571 

Chantemesse,  424 

Chantemesse  and  Widal,  412,  418 

Chapin,  562 

Charrin,  577,  578 

Charrin  and  Roger,  228 

Chauveau,  195,  574 

Christen,  67,  69 

Christmas,  385 

Chudiakow,  27 

Churchman,  140 

Churchman  and  Michael,  140 

Citron,  273,  294 

Clairmont,  449 

Clarke,  658 

Class,  676 

Clegg,  506 

Clough,  365 

Cobbett,  524 

COHENDY,    717 

Cohn,  5,  36,  38 

Coleman  and  Buxton,  405 

Coles,  504 

Conn,  415,  702,  703,  710,  712 

CoNRADi,  137,  405,  443,  704 

CONRADI    AND    DRIGALSKI,    134 

Cornet,  342,  487 


INDEX  OF  AUTHORS 


801 


CoRNiL  AND  Babes,  669 

Councilman,  658 

Councilman,    Malloby  and   Wright, 

374,  376,  377 
courmont  and  doyen,  463 
Craig,  787 

Craig  and  Nichols,  604 
Cramer,  21 

Creel,  Facet  and  Wbightson,  92 
Cbeite,  460 
Crowell,    561 
Curtis,  631 

CUSHING,  431 

cushino  and  livingood,  392 

Dale,  303 

D'Arsonville  AND  Charrin,  66 

Davaine,  6,  563 

Davis,  343 

Dean,  283,  510 

Debrand,  461 

Delezenne,  201 

Deneke,  591 

Denys,  346,  490,  492,  497 

Denys  and  Leclef,  281,  346 

Denys  and  Marchand,  346 

Denys  and  Van  de  Velde,  331 

De  Schweinitz  and  Dorset,  22,  490 

Deslongchamps,  326 

De  Toma,  486 

Deutsch  and  Feistmantel,  292 

DiEUDONN^,  64,  216,  566,  584 

Dochez,  365 

DocHEZ  AND  Avery,  364 

Dochez  and  Gillespie,  363 

DoERR,  186,  443 

doerr  and  russ,  302 

Donath  and  Landsteiner,  249 

DoNiTZ,  188,  218 

DoPTER,  379 

doutrelepont,  504 

Dreyer  and  Madsen,  209,  215 

Drigalski,  695 

Drigalski  and  Conradi,  406 

Dubarre  and  Terre,  501 

Dubois  and  Olmstead,  379 

Duclaux,  714 

DUCREY,    548 


V.  Dungern,  201,  242,  411 

Dunham,  386,  584 

Durham,  428,  434 

Durham  and  Myers,  669 

DuscH,  4 

dutton  and  todd,  608 

Duval,  506 

Duval  and  Wellman,  506 

DWYER,    304 

Eberth,  399 

Ehrenberg,  2 

Ehrlich,  7,  104,  187,  193,  199,  203,  204, 

205,  206,  209,  210,  213,  238,  464,  481 
Ehrlich,   Kossel  and   Wassermann, 

205 
Ehrlich   and  Morgenroth,   226,    242, 

243,  248 
Ehrlich  and  Sachs,  243,  244 
Eichstedt,  639 
Eisenberg,  39,  580 
Eisenberg  and  Volk,  420 

ElSENBREY,    303 

v.  elsler  and  pribram,  459 

Ejkmann,  47,  49 

Elser,  377 

Elser  and  Huntoon,  376,  378,  379,  387 

Elsner,  408 

Endo,  134 

Engelmann,  27,  61 

Eppinger,  573,  620 

Epstein,  77,  178,  619 

Ernst,  11,  579,  707 

EscHERiCH,  343,  389,  394,  453,  515,  700, 

716        • 
V.  Esmarch,  68,  147,  149 
Etre,  166,  550,  705 
Evans  and  Russell,  92 
Ewing,  596,  658 

Faget,  92 

Fantham  and  Stevens,  754 
Farnet,  413 
Faure-Beaulieu,  384 
Fehleisen,  336,   341,  342 
Fermi  and  Pebnossi,  462 
Ferran,  28,  195,  458,  590 
Ferri  and  Celli,  64 


802 


INDEX  OF  AUTHORS 


FicKEB,  62,  231,  423 

Field,  677 

Finger,  530,  535 

Finger  and  Landsteineb,  603 

FiNKELSTEIN,    579 

Finkler  and  Prior,  591 

FiNLAY,   670 

Firth  and  Horrocks,  687 

Fischer  and  Proskauer,  88 

Fisher,  9,  56,  59,  325 

Fisher,  A.,  10,  19,  23,  24 

Fitzgerald,  450 

Flexner,   197,  378,  413,  436,  444,  620 

Flexner  and  Jobling,  378 

Flexner  and  Lewis,  665 

Flexner  and  Noguchi,  462 

Flugge,  84,  85,  198,  325,  385,  586 

FoA,  359 

FoA  AND  Carbone,  362 

V.  FoDOR,  198,  224 

Foges,  460 

FONTANA,    598 

Foote,  415 

FORNEACA,  334 

FoRSTER,  F.,  32,  586 
Fracastor,  2 

Frankel,  a.,  353,  355,  360,  361,  404, 
412,  421,  447,  472,  504,  570,  619,  686 
Franzott,  326 
Freudenreich,  714 
Friedberger,  304 
Friedbeeger  ANT)  Hartoch,  303 
Friedlander,  352,  447 
Friedmann,  501 
V.  Frisch,  451 

Frosch  and  Kolle,  339,  368 
fuhbmann,  44 
Fuller,  692 
furbringer,  88 

Gabbet,  105,  481 

Gaffky,  333,  399,  400,  469 

Gaffky,  Pfeiffer,  Sticker,  and  Dieu- 

DONN^,   561 
Galtter,  486,  535 
Gamaleia,  590 
Garre,  327 
Gartner,  429 


Gavino  and  Girard,  677 
Gay,  244,  245,  427,  446 
Gay  and  Adler,  302 
Gay  and  Claypole,  405 
Gay  and  Southard,  300,  301 
Gengou,  240,  245,  273 
Geppert,  86 
Gessard,  577,  579 
Gheorghiewski,  581 
Ghon  and  Pfeiffer,  376,  386 
Ghon  and  Preyss,  539 
Gibson,  219 
Gibson  and  Collins,  219 

GlEMSA,    108 

Gilbert,  430 
Gilchrist,  631 
Gillespie,  363 
Globig,  32 
Goldberger,  677 

GOLDBERGER,    WiLLIAMS    AND    HaCHTEL, 

522 
Goodwin  and  v.  Sholly,  376 
Gordon,  348 

GORGAS,  670 

gottschlich,  23,  557 

gougerot,  644 

Gram,  102 

Gran,  49 

Grancher  and  Ledoux-Lebard,  486 

Grassberger,  539 

Grawitz,  640 

Grekoff,  718 

Gruber,  67,  78,  421 

Gruber  and  Durham,  200,  228,  234 

Gruby,  643 

Grund,  584 

Grunhagen,  657 

GuARNiERi,  357,  358,  359,  657 

Guerin,  662 

GUITERAS,    673 

GUMPRECHT,    463 

GUNTHER,   352 

GwYN,   431 

Haas,  66 
Hachtel,  522 
Haendel,  363 
Haenlein,  720 


INDEX  OF  AUTHORS 


803 


Hafpkine,  590 

Hahn,  190,  191,  416,  492 

Hallier,  7 

Hammabsten,  46 

Hammebschlag,  22,  490 

Hanel,  326 

Hankin  and  Leumann,  20 

Hankin  and  Wesbbook,  168 

Hansemann,  501 

Hansen,  505,  631 

Habding  and  Ostenbebg,  135,  433 

Habbison,  425 

Habbison  and  Galtieb,  715 

Habtman,  340 

Habtoch,  303 

Hauseb,  151,  454 

Heim,  704 

Heiman,  381,  384 

Heinemann,  701,  705 

Hektoen,  283,  675 

Hektoen  and  Ruedigeb,  282,  283 

Hellmich,  22 

Hellbiegel  and  Wilfabth,  55,  56 

Henle,  3 

Henbijean,  459 

Hebicoubt  and  Richet,  296 

Hebteb,  177,  394,  474 

Hesse,  27,  133,  485,  705 

von  Hibleb,  Kolle  and  Wassebmann, 

465 
Hilbebt,  31,  521 
Hill,  86,  94,  694 
Hibsch,  554,  587 
Hibschbebgeb,  707 
Hiss,   13,  98,   132,  251,  289,  337,  338, 

339,  340,  347,  348,  350,  354,  355,  356, 

357,  363,  365,  369,  370,  407,  408,  411, 

439,  440 
Hiss  and  Atkinson,  198,  219 
Hiss  and  Russell,  438 
Hiss  and  Zinsseb,  291,  367,  379 
HoFEB,  452 
Hoffman,  233 

Hoffman n-Wellenhoff,  523 
HoGYES,  652,  655,  656 
HoLST,  430 
Home,  675 
Hopkins,  604 


Hopkins  and  Lang,  349 
Hobton-Smith,  407,  411 
Houston,  686,  696 

HOWABD,    451 

Howabd  and  Perkins,  350,  369 
HuBEB,  538,  540 

HUDDLESTON,    662 

HuEPPE,  551 

HuEPPE  AND  Wood,  575 

Huntemulleb,  691 

HUNTEB,    198 

Isbael,  622 
IwANOw,  569 

Jackson,  137,  429,  453 

Jackson  and  Melia,  133,  138 

Jageb,  373,  451 

Jennee,  108,  193,  659 

Joachim,  198 

Johnson,  243 

Johnston,  404 

Jones,  75 

Joos,  233 

Jobdan  and  Heinemann,  700 

JOBDAN   AND  IbONS,    692 

Jobdan,  Russell  and  Zeit,  414 

JOBGENSEN  AND  MADSEN,  233 

Kamen,  394,  534 

Kappes,  21 

Kablinsky,  411 

Kelly,  343 

Kempneb,  li99 

Kempneb  and  Pollock,  478 

Kempneb  and  Schepilewsky,  215 

Kendall,  135 

Kibcheb,  1 

KisTEB  AND  Wolff,  237 

Kitasato,  456,  459,  462,  465,  555,  559 

KiTASATO  AND  WeyL,  27 

Kitt,  468 

KiTT  AND  Mayr,  554 

Klebs,  7,  352,  493,  512,  669 

Klein,  176,  404,  424,  480 

Klempeber,  503 

Klempebeb,  G.  and  F.,  362 

Klingmulleb  and  Baebmann,  604 


804 


^S 


INDEX  OF  AUTHORS 


Klotz,  718 
Knapp,  524,  542 
Knoepfelmacher,  665 
Knorr,  205,  295,  340 

KOBERT  AND   StILLMARCK,  204 

Koch,  7,  63,  73,  77,  84,  86,  89,  321,  335, 
352,  404,  469,  479,  483,  490,  491,  492, 
493,  496,  497,  498,  5^2,  563,  566,  569, 
570,  582,  587,  609,  610,  696 
Koch  and  Petruschky,  345 
Koch  and  Rabinovitsch,  500 
Koch  and  Wolffhugel,  65,  570 
Koch,  Gaffky,  and  Loeffler,  65,  564 
KoLLE,  465 

KoLLE  AND  Hetsch,  593 
KoLLE    AND     Wasseemann,     189,     190, 
191,  378,  502 

KOLMER,  659 

KoRN,  502 

KORSCHUN,   291 

KossEL,  204,  532 

KOSSEL    AND    OVERBECK,    551 

KossEL,  Weber,  and  Heuss,  499,  500, 

708 
KoTJAR,  628 
Kraunhals,  569 
Kraus,    186,   200,   235,   237,   328,   329, 

423,  443,  446,  589,  590 
Kraus  and  Doerr,  441,  444 
Kraus  and  Low,  232,  397 
Kraus  and  v.  Pirquet,  236 
Kraus  and  Stenitzer,  416 
Kresling,  532 
Kretz,  217 

Kronig  and  Paul,  74,  77,  78,  80 
Krumwiede,  136,  614 
Krumwiede  and  Pratt,  140 
Krumwiede,  Pratt,  and  Grund,  584 
Kruse,  292,  437,  444,  446,  540,  552 
Kurth,  431 
KiJSTER,  501 
Kutscher,  342,  431 
KuTSCHER  and  Meinicke,  433 

KUTSCHERT  AND   NeISSER,   524 

Kyes,  311 

Lachner-Sandoval,  618 
Landsteiner,*  201 


Landsteiner  AND  Jagic,  24^ 

Landsteiner  and  Levaditi,  665 

Landsteiner  and  Popper,  664 

Lang,  349 

Lanz  and   Tavel,  343 

Lassar,  602 

Leveran,  592 

Laws  and  Anderson,  695 

Leclainche  and  Vall^e,  467 

Ledderhose,  578 

Leeuwenhoek,  1 

Leichtenstern,  372 

Leishman,  281 

Lembke,  392 

Lentz,  438 

Lepierre,  377 

Le  Roy,  Des  Baeres,  and  Weinberg, 

368 
Lesage,  715 
Leuchs,  137 
Levaditi,  277,  280,  283 
Levaditi  and  Inmann,  283 
Levaditi  and  Manouelian,  599,  616 
Levaditi  and  Petresco,  596 
Lewith,  66 
LiBMAN,  339,  343,  431 

LiBMAN    AND    ROSENTHAL,    370 

LiBORius,  149 

LiEB,  304   • 

V.    Lingelsheim,  ■  328,    331,    337,    340, 

346,  348,  387 
LiNOSSIER  AND  Roux,  640 

Lister,  6 
LoEB,  324 
Loeffler,  14,  100,  110,  512,  520,  523, 

528,  530 
Loeffler  and  Frosch,  681 
Loeffler  and  Schutz,  528 
Loew,  25,  719 
Lohlein,  282 
Longcope,  432 
Lowen  stein,  501 

LUBARSCH,    190 

Lubbert,  *326 

LUERSEN   AND    KuHN,   715 
LUMSDEN,    415 

lustgarten,  503,  594 
Lynch,  Kenneth  M.,  739 


INDEX  OF  AUTHORS 


805 


Maassen,  53 

Macfadyen,  362 

Macfadyen  and  Rowland,  416 

MacNeal,  797 

MacNeal  and  Novy,  742,  750 

Madsen,  218,  521 

Mafucci,  480,  490,  500 

Mallory,  623,  676 

Malloby  and  Wright,  110,  112,  150, 

454 
Mann,  96 
Maragliano,  498 
Marbaix,  341,  342,  348 
Marchiafava  and  Celli,  371 
Marchoux  and  Salimbeni,  615 
Marchoux,    Salimbeni,   and    Simond, 

673 
Marchoux  and  Simond,  674 
Marie  and  Morax,  463 
Marinesco,  478 
Marmier,  344 
Marmorek,  344,  498 
Marschal,  371 
Martin,  L.,  468,  521 
Martin  and  Cherry,  204 
Martini  and  Lentz,  438 
Marx,  347 
Massol,  717 
McClintic,  80 
McCoy,  510,  559,  560,  562 
McCoy  and  Chapin,  562 
Mennes,  362,  365 
Mesnil,  232 
Messea,  15 
Metchnikoff,  188,  200,  201,  224,  225, 

228,  232,  275,  588,  603,  716 
Metchnikoff  and  Besredka,  426 
Metchnikoff  and  Roux,  601 
Metchnikoff,  Roux,  and  Salimbeni, 

689 
Meyer,   187 

Meyer  and  Ransom,  463 
Michaelis,  96,  254 
Michel,  516 
Michelson,  619 

MiGNESCO,   63 

MiGULA,  12,  36,  325 
Mikulicz,  452 


Miller,  411,  494,  706 
MiNCHiN  AND  Thompson,  744 
MiQUEL,  32,  690 
MiTcijELL,  J.  p.,  167 

MOELLER,    98,    501 

MoHLER,  495,  681,  707,  708,  711,  715 
Moll,  198 
Moltschanoff,  385 

MOMONT,   570 

Monti,  658 

MoRAX,  545 

Morax  and  Marie,  462 

Morgan,  404,  431 

Morgenroth,  202 

Morgenroth  and  Sachs,  266 

Moro,  492 

MorpurgO,  189 

MosER,  676 

MouTON,  275 

Moxter,  242 

Much,  482 

MiJHLENS,  600,  603 

Muhlschlegel,  17 

MiJLLER,  334,  504,  706 

MULLER,   Fr.,  325 

MuLLER,  Otto  Friedbich,  2 

MtJLLER,  p.  Th.,  244,  254,  255 

Mulzer,  595,  602 

MuNTZ,  25 

Muntz  and  Schlossing,  57 

Myers,  215,  253 

Naegeli,  38 

Nakanishi,  10,  11,  16,  17 

Nastjukoff,  539 

Neal  and  Schweitzer,  379 

Needham,  4 

Negri,  648 

Neisser,  107,  329,  380,  505,  514 

Neisser,     Baebmann,     and     Halber- 

STADTER,    615 

Neisser  and  Friedmann,  241 
Neisser  and  Sachs,  247,  273 
Neisser  and  Shiga,  443 
Neisser    and    Wechsberg,    201,    246, 

329,  330,  331 
Nencki  and  Scheffer,  21 
Neufeld,  345,  363,  365,  370,  412 


806 


INDEX  OF  AUTHORS 


Neufeld  and  Haendel,  363 
Neufeld  and  Hune,  283 
Neufeld  and  Rimpau,  282,  365 
Neufeld  and  Topfer,  283 
Neumann,  411,  579 
Nichols,  604 

NiCOLAIER,  456 

Nicoll,  464 

Nicolle,  233,  298,  300,  506,  602 

Nielsen,  481 

NiKATi  AND  Reitsch,  587 

Nikola YSEN,  385 

Nissen,  76 

NocARD,  430,  500,  530,  620 

NocARD  AND  Roux,  10,  480,  500 

NoGUCHi,  243,  264,  266,  269,  600,  601, 

604,  616,  617,  651,  794 
NoRRis,  237,  253,  424 
NoRRis  AND  Larkin,  620,  621 
NoRRis,    Pappenheimer,    and    Flour- 

NOY,  606 

North,   718 

NOTTER   AND    FiRTH,   706 

NovY  AND  Freer,  64 
NovY  AND  Knapp,  592,  606,  610 
NovY  AND  MacNeal,  742,  750 
Nuttall,  198,  224,  237,  242,  253,  560 
Nuttall  and  Thierfelder,  392 

Obermeier,  6,  605 

Obermuller,  711 

Ogston,  321,  335 

Ohno,  439,  446 

Olitsky,  678 

Olmstead  and  Dubois,  379 

Omelianski,  49,  58 

Omeltschenko,  79 

Ophuls,  631,  632 

Oppenheimer,  43,  47 

ostenberg,  135 

Ottenberg,  261 

Otto,  297,  298,  300,  669,  674 

Ottolenghi,  359 

Overton,  187 

Palmer,  349 

Pane,   232,   362,  366 

Papasotiriu,  391 


Pappenheim,  106,  483 

Park,  89,  216,  217,  412,  700 

Park  and  Carey,  439 

Park  AND  Dunham,  438 

Park  and  Holt,  705,  709 

Park  and  Krumwiede,  488 

Park  and  Nicoll,  464 

Park  and  Throne,  220 

Park  and  Williams,  521 

Park  and  Zingher,  527 

Parodi,  602 

Pasquale,  338,  591 

Passet,  326,  337 

Pasteur,  5,  41,  189,  192,  193,  194,  196, 

321,  352,  468,  552,  553,  573,  631,  647 
Pasteur  and  Chamberland,  122 
Pasteur,    Chamberland,    and    Roux, 

194,  571 
Paul,  659,  660,  661,  663 
Peabody  and  Pratt,  137,  409 
Pearce,  202 

Pearce  and  Eisenbrey,  303 
Penfold,  39 
Perez,  452 
Perkins,   448 
Perrone,  339,  344 
Petri,  502 
Petroff,  484 
Petruschky,   342,  404,   411,  427,   618, 

620,  679 
Petterson,  291 
Pfaundler,  231,  232,  234 
Pfeffer,  54,  56 
Pfeiffer,  195,  199,  230,  255,  279,  416, 

536,  537,  541,  589,  657 
Pfeiffer  and  Beck,  536,  541 
Pfeiffer  and  Friedberger,  242 
Pfeiffer  and  Isaeff,  199,  224 
Pfeiffer  and  Kolle,  231,  418,  425 
Pfeiffer  and  Nocht,  588,  591 
Pfeiffer  and  Wassermann,  589 
Pfluger,  59 
Pfuhl,  455,  540 
Pierrallini,  276 
V.  Pirquet,  302,  494 
V.  Pirquet  and  Schick,  296,  301 
Pitt,  14 
Plaut,  611,  642 


INDEX  OF  AUTHORS 


807 


Plenctz,  2 
Plotz,  678 
Plotz,  Olitsky,  and  Baehb,  678 

POELS    AND    DhONT,    430 

poels  and  nolen,  368 
pollender,  6 
Poor,  651 

PORGES,    13 

PORTIER  AND   RiCHET,   296 

POTT,  706 

Pratt,  140,  413,  584 

Pbescott,  391,  696,  697 

Preuseb,  532 

Proscheb,  331,  332 

Proskauer  and  Beck,  29 

Prudden,  404,  490 

Prudden  and  Hodenpyl,  490 

Pryor,  487 

Quincke,  716 

Rabinovitsch,  502,  510,  634,  678,  711 

Radziewsky,  363 

Ransom,  463 

Ransome  and  Fullerton,  88 

Ravenel,  500,  570 

V.  Recklinghausen,  7 

Redtenbacher,  407 

Reed,  670 

Reed  and  Carroll,  430 

Reed,  Carroll,  and  Agramonte,  670 

Reed,  Carroll,  Agramonte,   and  La- 

ZEAR,  669,  670,  674 
Remlinger,  651 
Richardson,  64,  411,  412 
RiCHET,  296,  301 
Richet  and  Hericourt,  197,  331 

RiCKETTS,  633 
RiCKETTS  AND  WiLDER,    677 
RiDEAL  AND  WALFER,  80 
RiEDER,    64 

RiMPAU,  365 

RiNDFLEISCH,    7 

RlXFORD  AND  GiLCHBIST,  632 

Roger,  344 

ROHNER,  579 

RoMER,  476 


ROSENAU  AND  ANDERSON,  222,  297,  298, 

519 
RoSENAU  AND  McCoY,  699 
RoSENAU,  LUMSDEN  AND  CaSTLE,  415 
ROSENBACH,  321,  337 

rosenberger,  489 
Rosenthal,  441 
RosENOW,  39,  343,  370 
Ross  AND  Milne,  609 
RosT,  506,  509 

ROTHBERGER,   403 

Roux,  64,  149,  203 

Roux  AND  Chamberland,  471 

Roux  AND  NOCARD,   533 

Roux  AND  Yersin,  107,  512,  520,  522 

RUBNER,  24 

RUPPEL,  22,  491 
Russell,  138,  426,  797 
Russell  and  Fuller,  695 

Sabouraud,  643 
Sacharoff,  616 
Sachs,  188,  199,  202 
Sachse,  54 
Sahli,  493 
Salmon,  552 
Salmon  and  Smith,  196 
Sanarelli,  669 
Sanfelice,  471,  632 
Sauerbeck,  293 
Saul,  77 
Savage,  231 

SCHAEFFEB  AND  StEINSCHNEIDER,  384 
SCHAFER,    30 

Schattenfroh,  291,  328 

SCHATTENFROH    AND    GrASBERGER,    702 

schaudinn,  592 

schaudinn  and  hoffmann,  594 

Schell  and  Fischer,  486 

SCHENCK,  644 
SCHERESCHEWSKY,  599 
SCHERING,  91 

SCHEUERLEN  AND  SpIRO,  74,  78 
SCHILD,  392 

Schimmelbusch,  327 
schlesinger,  345 
Schlossmann,  90 
Schneider,  325 


808 


INDEX  OF  AUTHORS 


SCHNITZLEB,  455 
SCHOLTZ,   380 
SCHOTTELIUS,   392 

ScHOTTMiJLLER,  337,  344,  348,  350,  370, 
405,  431,  706 

SCHREIBER,   546 

SCHROEDER,  4 

SCHBOEDER  AND  COLTON,  707 

SCHROETER,    60 

SCHUDER,   414,    703 

SCHiJLLER,   669 

SCHULTZ,   303 

SCHULZE,   4 

ScHUTZ,  341,  368 

ScHUTZE,  202,  236 

Schwann,  3,  4 

Schweitzer  and  Neal,  379 

ScLAVO,  357,  574 

Sedgwick  and  Batchelder,  700 

Sharnosky,  103 

Shattock,  22 

Shiga,  435,  438,  442,  444,  446 

SlEDENTOPF,  597 

Signorelli,  140 

SiLBERSCHMIDT,   455 

Simon  and  Lamar,  286 

Simon,  Lamar,  and  Bispham,  286 

SiMOND,  560 

Simpson,  705 

Simpson  and  Hewlett,  80 

Smith,  101,  499,  500,  708 

Smith,  Graham,  524 

Smith,  Herbert  E.,  704 

Smith,  Th.,  27,  34,  297,  485,  486,  498, 

499,521 
Smith,  Th.,  and  Kilbourne,  19i5 
Smith,  Th.,  and  Moore,  430 
Smith,  Th.,  Brown  and  Walker,  458 
Sobernheim,  563,  574 
Sobernheim  and  Tomasczewski,  595 
Solitsky  and  Zingher,  519 
Sommerville,  80 
Southard,  301 
Spallanazani,  Abb6,  4 
Spengler,  493 
Spilker  and  Gottstein,  65 
Spitzer,  595 
Spronck,  437 


Spronk,  521 

Stefansky,  510 

Steinhardt,  Dr.,  651 

Stephens,  787 

Stern,  237,  421 

Stern  and  Korte,  259,  418 

Sternberg,  33,  325,  340,  352,  359,  516, 

669 
Stevens  and  Myer,  754 
Stevens  and  Myers,  204 
Sticker,  508,  644 
Stokes,  343 

Stokes  and  Weggefarth,  706 
Strauss,  532 

Strauss  and  Gamaleia,  490,  500 
Strauss  and  Huntoon,  652 
Strelitz,  519 
Stricht,  478 
Strong,  L.  W.,  449,  561 
Strong  and  Musgrave,  436 
Strong,  Teague,  and  Barber,  561 
Strong,  Teague  and  Crowell,   561 
suchsland,  719 
Surmont,  201 

SURMONT  AND  ArNOULD,  570 

Suzuki  and  Takaki,  489 

Tacke,  54 

Talamon,  353 

Tarozzi,  460 

Tavel  and  Krumbein,  368 

Taylor,  645 

Teague,  561 

Teague  and  Torrey,  383 

Tedesco,  540,  541 

Terin,  327 

Thayer,  669 

Thomas,  668 

Thompson  and  Minchin,  744 

TiDSWELL,  510 

TissiER,  715 

TissiER  AND  Mabtelly,  392,  716 

TizzoNi,  460 

Todd,  329,  443 

Tokishige,  632 

Tomasczewski,  596 

ToRiNi,  47 

Torrey,  383,  385 


INDEX  OF  AUTHORS 


809 


TOTSUKA,  419 

ToussAiNT,  194,  571 
Trask,  704 
Teillat,  89 
tschistovitch,  236 
tsiklinski,  32 

TuifNICLIFF,  612,  613 
TUBNBULL,  610 
TWOBT,  39 

Uhlenhuth,  237 

UHLENHUTH  AND  MULZEB,  602 

Ullmann,  384 

Unna,  639 

UscHiNSKY,  28,  126,  522 

Vagedes,  499 

Vaillard  and  Dopter,  443 

Vaillard  and  Rouget,  459 

Vaillard  and  Vincent,  462 

Valleri-Radot,  4 

Vallet,  695 

Van  der  Loeff,  657 

Van  de  Velde,  329,  330,  342,  346 

Van  Ermengem,  14,  101,  430,  476 

Van  Gehuchten,  648 

Van  Gieson,  649 

Vaughan,  39,  298,  304,  417 

Vaughan  and  Wheeler,  301 

Vedder  and  Duval,  438 

Veeder,  415 

Veillon,  333 

Di  Vestea  and  Zagabi,  647 

ViGNAL,  619 
ViLLEMIN,  479 

Vincent,  611 

VOGES,  29 

Von  DEM  Borne,  615 

VOTTALER,  457 

Wadsworth,  99,  249,  355,  357,  359,  360, 

363,  365 
Wagmann,  703 
Waldeyer,  7 
Walker,  419 
Ward,  64,  703 
Washburn,  365 


Wassermann,  195,  199,  204,  236,  243, 

382,  385,  465,  579,  580,  604 
Wassermann  and  Bruck,  247,  263 
Wassermann  and  Citron,  293 
Wassermann,    Neisser,    and    Bruck, 

263 
Wassermann  and  Proskaueb,  522    ' 
Wassermann  and  Schutze,  237 
Wassermann  and  Takaki,    188,  214, 

279,  463 
Wassilieff,  532 
Watson,  A.  E.,  749 
Weber,  501 
Wechsberg,  243 
Weeks,  542 
Weeny,  419 
Wegele,  718 
Weichselbaum,  333,  353,  372,  376,  386, 

447,  450,  540 
Weichselbaum  and  Muller,  542 
Weigert,  7,  111,  214,  481 
Weil,  303 

Weill-Hall  and  Lemaire,  300 
Weis,  a.  H.,  328 
Weiss,  482 

Welch,  98,  129,  355,  357,  395,  472 
Welch  and  Blachstein,  404 
Welch  and  Flexner,  473 
Welch  and  Nuttall,  177,  471 
Wells,  302 
Wenyon,  758 
Wernicke,  561 
Wertheim,  381 
Wesenberg,  455 

Westphal  and  Uhlenhuth,  508 
Weyl,  22,  481,  490 
Wherry,  510 

WiCKMAN,  664 

WiDAL,  421 

WiDAL  AND  NoBECOURT,  430 

WiDAL  AND  SiCARD,  421 

WiLCKENS,   704 

WiLDE,  451,  452,  453 
Wilder,  677 
Williams,  522,  533 
Williams  and  Lowden,  650 
Willson,  695 
Wilson,  557 


810 


INDEX  OF  AUTHORS 


WiLTSCHOUB,    407 
WiNOGRADSKY,    14,    54,    57 

WiNSLOw,  343 

WiNSLOw  AND  Palmer,  340 

Wladimiroff,  533 

WOLBACH,  681 

WOLBACH  AND   EeNST,  499 

Wolff,  306 

Wolff  and  Israel,  624 
Wolff-Eisner,  301,  404 
wolffhugel,  89 
Wollstein,  541,  544,  545 
Wood,  95,  109,  359,  720 

WORONIN,   55 

Wright,   108,    150,   153,   195,  286,  288, 

425,  623,  624,  627,  758 
Wright  and  Douglas,  281,  282 


Wright  and  Lamb,  550 
Wrightson,  92 
Wu  Lien  Teh,  560 

Yebsin,  565 

Yebsin,  Calmette  and  Roux,  561 

Zeit,  64 

Zettnow,  10,  11,  12,  606 

ZiEHL,  97,   105,  481 

Zingher  and  Park,  527 

Zingher  and  Soletsky,  519 

Zinsser,    16,    155,   243,   291,   411,  413, 

442,  519,  524,  631 
Zinsser  and  Gary,  511 
Zinsser  and  Hopkins,  604 
Zinsser,  Lieb  and  Dwyer,  304 


INDEX  OF  SUBJECTS 


Abbott's    method    of   staining    spores, 

97 
Abscess  of  liver,  due  to  amebic  dysen- 
tery, 726 
Absorption  method  in  study  of  agglu- 
tination reaction,  234 
Achorion  Schoenleinii,  642 

cultivation  of,  642 

morphology  of,  642 

varieties  of,  642 
Acid  formation  by  bacteria,  166 
Acid-fast  bacteria,  stains  for,  104 
Acquired  immunity,  192 

definition  of,  192 
Actinomyces,    622 

cultivation  of,  623 

discovery  of,  in  cattle,  622 
in  man,  622 

morphology  of,   619,  622 

pathogenicity   of,    625 
in  animals,  625 
in  man,   625 

parts  of  body  infected  in,  625 

staining  of,  623 

varieties  of,  626 
Actinomycosis,  625 
Active  immunity.     See  Immunity 
Aerobic  organisms,  facultative,  26 

obligatory,    25 

non-infectiousness   of,    183 

respiratory   processes   of,   27 
Aestivo-autumnal    fever,   diagnosis    of, 
with  fresh  blood,  775 

parasite   of,    771 

symptoms  of,  776 
Agar  for  colon-typhoid  differentiation, 
brilliant  green,  136 

MacConkey's  bile  salt,  138 

Russell's  double  sugar,  138 


Agar  for  culture  media,  127 
lactose-litmus,  129 
meat  extract,  127 
meat  infusion,    128 
Agar    slants,   cultivation   of   anaerobic 

bacteria  on,  153 
Agglutination  reaction,  228 
between    agglutinin    and    agglutinin- 

stimulating  substances,  233 
clinical  diagnosis  by,  in  typhoid,  229 
concentrated  agglutinin  in,  235 
differentiation  of  bacterial  species  by, 
229 
upon  dead  bacteria,  231 
diluted  agglutinins  in,  235 
group    agglutination    in,    234 
immune  or  chief  agglutinin  in,  234 

upon  living  bacteria,  231 
macroscopical  observation  of,  230 
for  bacterial  differentiation,  231 
major  agglutinin  in,  234 
microscopical  observation  of,  229 

for  clinicial  diagnosis,  231 
minor  agglutinins  in,  234 
nature   of,    228 
of  capsulated  bacteria,   13 
partial  agglutinins  in,  234 

absorption    method    in    study    of, 
234 
proagglutinoid  zone  in,  235 
proagglutinoids   in,   235 
pseudo-clumping  in,  231 
specificity  of,   234 
"thread-reaction"  in,  231 
Agglutination  tests,  technique  of,  251 
macroscopic,  252 
microscopic,  252 
Agglutinins,  200,  228 
action  of,  240 
811 


812 


INDEX  OF  SUBJECTS 


Agglutinins,    agglutinin-stimulating 
substances  and,  233 
quantitative  relations  between,  233 
reaction    between,    233. 
bactericidal      substances      compared 

with,  231 
cell  receptors  in,  238 
concentrated,  failure  of,  to  produce 

agglutination,  235 
diluted,  agglutination  reaction  with, 

235 
effect  of  heat  on,  232 
experimentation   with,   231 
in    agglutination    reaction,    chief    or 
immune,  234 
major,  234 
minor,   234 
partial,   234 
in  serum  of  glanders,  535 
in      staphylococcus      immune     sera, 

331 
nature  of,  231 
normal,  232 
partial   absorption  method  in   study 

of,  234 
production  of,  232 

in  sera  of  animals,  by  injection  of 
bacteria,  233 
of  culture  extracts,  233 
time  of,  233 
reaction  of.    See  Agglutination  reac- 
tion 
specificity  of,  234 
structure  of   (Ehrlich),  238 
theoretical     considerations     concern- 
ing, 238 
"thread-reaction"  in,  231 
Agglutinogen,  233 
"Aggressin  theory"  of  Bail,  292 
arguments  for,  292,  293 
basic  principles  of,  292 
opposition  to,  293,  294,  314 
Aggressins    (Bail's  theory),  293,  313 
action  of,   292 
immunization  with,  293 
nature  of,  293 
occurrence  of,  293 
Air,  bacteria  in,  673 


Air,  dryness  and  high  winds  favorable 
to  increase  of,  683 
estimation  of  numbers  of,  684 
occurrence  of,  in  inhabited  places, 

683 
scarcity  of,  in  places  high  above 

earth,  684 
settling  of,  with  rain,  snow,  etc., 
684 
infectious  material  carried  by,  684 
Alcohol,  as  fixative  in  staining,  110 
Alcoholic  fermentation,  51 
by  yeasts,  52 
in  milk,  684 
process  of,  51 
Alcohols  as  disinfectants,  77 
Alexin,    198 

action  of,  in  blood  serum,  224,  225 
Alkali  formation  by  bacteria,  166 
Allantiasis,  477 
Allergic,  302,  304 

Amboceptor    and    complement,    quanti- 
tative relationship  between,  246 
Amboceptors,  filtration  of,  245 

multiplicity  of,  in  normal  sera,  242 
Ameba  proteus,  723 
Amebae,    characterization    and   develop- 
ment of,  723 
free-living  varieties   of,   ameba   pro- 
teus, 723 
"limex  amebae"   (Volkampfia),  723 
parasitic  varieties  of,  endameba  coli, 
724,   734 
endameba  gingivalis,  724,  736 
endameba  histolytica,  724,  725 
pathogenic  history  of,  725 
pathogenicity  of,  726 
Amebic    dysentery,    complications     of, 
726 
diagnosis  of,   727 

diff'erentiated    from   bacillary  dysen- 
tery, 726 
geographical  distribution  of,  727 
history  of,  726 
prevention  of,  733 
symptoms  of,  726 
treatment  of,  732 
Ameboid  organisms,  amebae,  723 


INDEX  OF  SUBJECTS 


813 


Ameboid  organisms,  endomeba,  723,  724 
Amylase,   48 
action  of,  49 
occurrence  of,  49 
Amylolytic  ferment.     See  Amylase 
Anaerobic  blood  cultures,  180 
Anaerobic  cultivation   of  bacteria,   148 
use  of  sterile  tissue  as  an  aid  in,  156 
See  also  Cultivation  of  bacteria 
Anaerobic  organisms,  facultative,  26 
Gram-positive,   in   feces,    177 
infectiousness  of,  183 
non-invasion    of    blood     stream     by, 

183 
obligatory,  26 
respiratory  processes  of,  26 
Anaphylatoxin,  304 
Anaphylaxis,  295 

autopsy  findings  in,  299 

definition  of,  297 

desensitization  to,  300 

experimentation  in,  early,  295 

immunity  after,  299 

in    diphtheria    antitoxin    injections, 

296 
incubation  during,   299 
inherited,  300 

observations  in,  fundamental,  296 
by  Arthus,  297 
byBesredka,    298,    300 
by  Besredka  and  Steinhardt,  300 
by  Gay  and  Southard,  300 
by  Hericourt  and  Richet,  296 
by  Nicolle,  298,  300 
by  Otto,  297,  300 
by  Portier  and  Richet,  296 
by  Rosenau  and  Anderson,  297, 

298 
by  Th.  Smith,  297 
by  Vaughan  and  Wheeler,  298 
by  Weill-Halle  and  Lemaire,  300 
passive,  300 
phenomena  of,  295 
"phenomenon  of  Arthus"  in,  297 
proteid   injections   in,    295 
mode  of  giving,  298 
quantity  of,  298 


Anaphylaxis,  symptoms  in,  299 
theories  concerning,  301 

based   on   Ehrlich's   receptor   over- 
production theory,  301 
of  Besredka,  301 
of  Doerr  and  Russ,  302 
of  Friedberger  and  Hartoch,  303 
of  Gay  and  Southard,  301 
of  Hamburger,  303 
of  Pearce  and  Eisenbrey,  303 
of  v.  Pirquet  and  Schick,  301 
of  Richet,  301 
of  Wolff-Eisner,  301 
Anilin   dyes,   influence  of,  upon  bacte- 
rial growth,   140 
Animal   alkaloids,   45 
Animal  experimentation,  169 
animals  used  in,   169 

cages  for,   173 
autopsies  in,   173 
inoculations  in,   170 
Anopheline.     See  Malarial  Mosquito 
Antagonism   of  bacteria,   30,  31 
Anthrax,  563 

bacterial   causation   of,   6 
occurrence  of,  563 
Anthrax  bacillus,  563 
action   of,   571 
bacilli  resembling,  575 

Bacillus   anthracoides,  575 
Bacillus  radicosus,  575 
Bacillus  subtilis,  575 
biology  of,  569 
cultivation  of,  566 
early  investigation  of,  563 
experimental   inoculation  with,   571 
immunization  against,  573 

active    (Pasteur's  method),  574 
attenuation  in,  573 
passive      ( Sobernheim's     method ) , 
574 
in   milk,   707 
infection  with,  572 
by  inhalation,  572 
cutaneous,  572 
pulmonary,  573 
spontaneous,  572 


814 


INDEX  OF  SUBJECTS 


Anthrax    bacillus,    infection    with, 
through  alimentary  canal,  573 
isolation  of,  565 
morphology  of,  564 
pathogenicity  of,  570 
prophylaxis  against,  573 
resistance  of,  570 
staining  of,  565 

susceptibility  of  animals  to,  570 
virulence  of,   570 
Anthrax,  symptomatic,  bacillus  of,  465 
cultivation  of,  465 
immunization  against,  468 

vaccines  used  in,  468 
morphology  and  staining  of,  465 
occurrence  of,  465 
pathogenicity  of,  466 

autopsy  findings  in,  467 
toxins  of,  467 
Anthropoid     apes,     blood     of,     distin- 
guished  from  human,  237 
Antiaggressins,  293 
Antiamboceptors,   243 
Antianaphylaxis,  299,  300 
Antibodies,  197 

experimentation    and    discovery    of, 
197 
agglutinins,  200 
alexin,   198 
antiferments,    202 
antitoxin,  198-199 
bacteriolysins,  200 
cytotoxins,  201 
precipitins,   200 
facts  and  theories  concerning,  242 
in   sera,    determination   of,   by   com- 
plement   fixation.     See    Comple- 
ment fixation 
to  gonococcus,  385 
Anticomplements,  243 
Antiferments,  202 

Antiformin,  formula  for,  as  given  by 
Rosenau,  483 
in  examination  of  sputum  for  tuber- 
cle bacilli,  483 
Antigen,  beef  heart  or  guinea-pig  heart 
muscle,  264 
cholesterinized,  264 


Antigen,  definition  of,  202 

determination  of,  by  complement  fix- 
ation, 271 

for   Wassermann  test,  263 
quantity  necessary  for,  265 

Noguchi's    acetone*  insoluble    lipoid, 
264 

"sensitization"    of,    241 

preparation  of,  264 
Antihemolytic  action,  243 
Antilab,  202 
Antilactase,  202 
Antileucocidin,  331 
Antipepsin,  202 

Antiricin,   discovery  and   experimenta- 
tion with,  204 
Antiseptics,  inhibition  strengths  of  va- 
rious, 84 

values  of,  determination  of,  80 
table  of,  80 
Antisera,   precipitating,  production  of, 

253 
Antistaphylolysin,  331 
Antisteapsin,  202 
Antistreptococcic   sera,  346 
Antitoxic  sera,  196 
Antitoxin,  198,  199 

diphtheria.   See  Diphtheria  antitoxin 

normal,   205 

production   of,   a.  final   test  between 
toxin  and  endotoxin,  187 

stability  of,  206 

tetanus.    See  Tetanus  antitoxin 

unit  of,  205 

valency  of,  for  toxin,  210 
Antivenin,   198 
Arthrospores,  16 
Ascospores,  630,  638 
Ash  in  bacterial  cell,  23 
Asiatic  cholera.     See  Cholera 
Aspergillus,  reproduction  in,  637 
Attenuated  cultures  in  active  immuni- 
zation, 193 
Autoclave,  71 

technical  details  of,  72 

Trillat's,  87 
Autointoxication,  gastrointestinal,  715 
bacteria  causing,  715 


INDEX  OF  SUBJECTS 


815 


Autointoxication,  gastrointestinal,  ex- 
perimental combating  of,  by 
acid-producing  bacilli,  716 
Metchnikoff's  treatment  of,  by 
means  of  Bacillus  bulgaricus, 
716-717 

Autolysins,  248 

Autopsies    of    infected    animals,    173 

Avian    tuberculosis,    bacillus    of.      See 
Tubercle  bacillus,  bacilli  related  to 

Babes-Ernst  granules,  11 
Bacilli,  diphtheroid,  525 
Bacilli    intermediate    between    typhoid- 
and  colon  organisms,  428 
bacterial  correlation  of,   431 

bacilli  of  colon-like  morphology  in, 

433 
bacilli  of  typhoid-like  morphology 

in,  433 
non-motile  bacilli  in,  433 
classification  of,  432 
differentiation  of,  from  typhoid  and 
colon  groups,  428 
by  cultural  characteristics,  428 
by  morphology,  428 
by  motility,  428 
"heterogenous  types"  of,  430 
hog-cholera  bacilli  in,  430 
meat-poisoning  bacilli  in,  429 
Bacillus  enteritidis   in,   429 
Bacillus  icteroides  in,  430 
Bacillus  Morseele  in,  430 
paratyphoid  bacilli  in,  430 
Bacillus  psittacosis   in,  430 
"Muller"  bacillus,  431 
paracolon  bacillus  in,  430 
"Seeman"  bacillus,  431 
pathogenicity  of,  431 
toxic  products  of,  431 
Bacillus,  37 

general  description  of,  9 
"heterogenous  types"  of,  430 
non-pathogenic  type  of,  430 
Bacillus  aerogenes  capsulatus,  471 
and  pernicious   anemia,   177,   474 
classification  of,  472 
isolation  of,  474 


Bacillus    aerogenes    capsulatus,    occur- 
rence of,  472,  474 
pathogenicity  of,  473 
Bacillus  anthracoides,  575 
Bacillus  avisepticus,  552 
Bacillus  botulinus,  475 
antitoxin  for,    199 
morphology  of,  476 
pathogenicity  of,  477 
staining  of,  476 
toxins    of,    478    . 
Bacillus  bulgaricus,  use  of,  by  Metch- 
nikoff  for  treatment  of  gastrointes- 
tinal autointoxication,  716-717 
Bacillus  butyricus,  502 
Bacillus  cloacae,  455 
Bacillus  coli  communior,  398 
Bacillus  coli  communis,  389 
agglutinins  for,  396 
in  immune  serum,  396 
in  normal  serum,  397 
bladder  diseases  due  to,  395 
cholera  infantum  attributed  to,   394 
cholera  nostras  attributed  to,  394 
cultivation   of,    390 
differentiation  of,  from  meat-poison- 
ing and  paratyphoid  bacilli,  428 
distribution  of,  391 
in  animals,  392 
in  feces,  177 
in  man,  392 
in  milk,  391 
in  nature,  391 
in  water,  696 
immunization  with,  395 
inflammatory  conditions  of  liver  and 

gall-bladder  attributed  to,  395 
isolation  of,  391 
morphology  of,  389 
pathogenicity  of,  393 
peritonitis   following  perforation  at- 
tributed to,  394 
septicemia  due  to,  394 
staining  of,  389 
toxic  products  of,  395 
varieties  of,  397 

Winckel's  disease  in  the  newborn  due 
to,  394 


816 


INDEX  OF  SUBJECTS 


u 


Bacillus    diphtherisB.      See    Diphtheria 

bacillus 
Bacillus  enteritidis,  discovery  and  char- 
acteristics of,  429 
Bacillus  fecalis  alkaligenes,  427 

diiferentiation  of,  from   typhoid  ba- 
cillus, 427 
in  feces,   177 
Bacillus    Hoffmanni.      See    Diphtheria 

bacillus,  bacilli   similar  to 
Bacillus  icteroides,  4^0 
Bacillus  influenzae.     See  Influenza  ba- 
cillus 
Bacillus  lactis  aerogenes,  453 
cultivation  of,  453 
morphology  of,  453 
occurrence  of,  453 

in  feces,  177 
pathogenicity  of,  454 
Bacillus  leprae.     See  Leprosy 
Bacillus  mallei,  528.    See  also  Glanders 
biological  characteristics  of,  530 
cultivation  of,  528 
immunity  against,  535 
morphology  of,  528 
pathogenicity  of,  530 

bacteriological  diagnosis  in,  532 
in  horses,  530 
in  man,  532 
nodules  in,  532 
spontaneous  infection  by,  530 
staining  of,  528 
toxin  of,  532 
action  of,  533 
diagnostic  use  of,  533 

directions  of  U.  S.  Government 
for,  534 
obtaining  and  preparation  of,  533 
Bacillus    melitensis.      See    Micrococcus 

melitensis 
Bacillus  mesentericus  in  feces,  177 
Bacillus  Morseele,  discovery  and  charac- 
teristics of,  430 
Bacillus  mucosus  capsulatus,  447 
association  of,  with  pneumonia,  450 
association    of,    with   other    diseases 

of  mucous  liningSj  450-451 
cultivation  of,  448 


Bacillus   mucosus   capsulatus,   cultural 
characteristics  of,  449 
Fitzgerald's  work  on  classification  of, 

450 
immunization  against,  451 
inoculation  of  animals  with,  451 
morphology  of,  447 
pathogenicity  of,  450 
staining  of,  448 
Bacillus  murisepticus,  542 
Bacillus  ozaenae,  452 
Bacillus  pestis.     See  Plague  bacillus 
Bacillus        prodigiosus,        quantitative 

chemical  analysis  of,  21 
Bacillus  proteus,  455 
Bacillus  proteus  vulgaris,  454,  455 

cultivation  of,  455 
Bacillus  proteus  mirabilis,  455 
Bacillus  proteus  zenkeri,  455 
Bacillus  proteus  zopfi,  455 
Bacillus  psittacosis,  430 
Bacillus  pyocyaneus,  577 
antitoxin  against,  580 

products  of,  199 
cultivation  of,  577 

favorable  conditions  for,  577 
pigment  in,  578 

fluorescent  variety  of,  579 
pyocyanin  in,  578 
immunization  against,  580 

filtrates  of  old  cultures  in,  580    • 
pyocyanase  in,  580 
true  toxin  in,  580 
morphology  of,  577 
occurrence  of,  in  lesions  and  inflam- 
matory affections,  579 
pathogenicity  of,  579 
staining  of,  577 

susceptibility  of  animals  to,  580 
toxins  of,  580 

leucocyte-destroying     ferment     in, 

581 
pyocyanase  in,  580 
pyocyanolysin  in,  580 
true,  580 

from  filtrates  of  old  cultures,  580 
virulence  of,  579 
Bacillus  radicicola^  55 


5 


INDEX  OF  SUBJECTS 


817 


Bacillus  radicosus,  575 
Bacillus  rhusiopathiae,  542 
Bacillus   smegmatis.     See  Smegma   ba- 
cillus 
Bacillus  subtilis,  575 
Bacillus  suisepticus,  553 
Bacillus  tetani.     See  Tetanus,  bacillus 

of 
Bacillus  tuberculosis.    See  Tubercle  ba- 
cillus 
Bacillus  typhi  abdominalis,  399 
Bacillus  typhi  murium,  430 
Bacillus  typhosus.     See  Typhoid  fever, 

bacillus  of 
Bacillus    xerosis.     See    Bacillus    diph- 

theriae,  bacilli  similar  to 
Bacteria.     See  also  Bacterial  cell 
acid  and  alkali  formation  by,  166 
acid-fast  stains  for,  104 
action  of,  in  the  body,  184 
anabolic    or    synthetic   activities    of, 
54 
in  root  tubercles,  55 
in  soil,  54 
animal  experimentation  with,  169 
antagonism  of,  30,  31 
biological  activities  of,  40,  164 
chemical  agents  injurious  to,  73.      See 

also  Disinfectants 
classes  of,  182 
bacilli,  9 
cocci,  9 
spirilla,  9 
classification  of,  35 

based  on  organs  of  motility,  15 
by  Bail  with  regard  to  aggressins, 

294 
by  Gram  stain,  104 
by  Migula,  36 
counting  of,   161 

in  milk,  710 
cultivation  of,  141 

by    anaerobic    methods,    148.     See 

also  Cultivation  of  bacteria 
inoculation  of  media  in,  141 
dead,  in  active  inmmunization,  193 
degenerative  forms  of,  20 
denitrifying,  52 


Bacteria,  destruction  of.     See  Destruc- 
tion of  bacteria 

differentiation    of,    by    fermentation, 
48 

dissimilar,  bacillus  cloacae,  455 
bacillus  mirabilis,  455 
bacillus  proteus  septicus,  455 
bacillus  proteus  vulgaris,  455 
bacillus  proteus  zenkeri,  455 
bacillus  proteus  zopfi,  455 
bacillus    probacillus    ligne    faciens 
septicus,  455 

enzymes  produced  by,  168 
diastatic,   169 
inverting,  169 
proteolytic,  168 

gas  formation  by,  164 

See  also  Gas  formed  by  bacteria 

Gram-negative,  104 

Gram-positive,   104 

in  air.     See  Air,  bacteria  in 

in  industries,  715 

in  milk.     See  Milk,  bacteria  in 

in  soil.     See  Soil,  bacteria  in 

in  tissues,  staining  of,  110 

in  water.     See  Water,  bacteria  in 

incubation  of,  156 

See  also  Incubation  of  cultures 

indol  production  by,  167 

invasive  power  of,  294 

isolation  of,  methods  of,  142 
from  milk,  710 

katabolic  activities  of,  41-53 

by  bacterial  enzymes  or  ferments, 
42 
varieties  of,  48 
by  denitrifying  bacteria,  52 
by  fat-splitting  enzymes,  47 
by  lab  enzymes,  46 
by- proteolytic  enzymes,  43 

liberation  of  energy  by,  58 

light  production  of,  59 

microscopic    study    of.      See    Micro- 
scopic study 

nitrifying.     See  Nitrifying  bac- 
teria 

nutrition  of,  25 

See  also  Nutrition  of  bacteria 


818 


INDEX  OF  SUBJECTS 


Bacteria,   occurrence  of,    in   the  body, 

181 
parasitic,  29 

definition  of,  182 
pathogenic,   182 
phenol  production  by,  167 
physical  agents  injurious  to,  62 
pigment  formation  by,  59 
protozoa  and,  differentiation  of,  1 
putrefactive,     quantitative    chemical 

analysis  of,  21 
reducing  powers  of,  167 
relation  of,  to  moisture,  34 

to  physical  environment,  31 

to  pressure,  34 

to  temperature,  31 
relationship  of,  to  other  plants,  35 
reproduction  of,  18 

rate  of,  18 

varieties  of,  18 
saprophytic,  29 

definition  of,  182 
size  of,  9 

staining  of,  methods  of.     See  Stain- 
ing of  bacteria 
sulphur.     See  Sulphur  bacteria 
symbiosis  of,  30,  31 
thermal  death  points  of,  33        ^ 
variations  in  forms  of,  19 
virulence  of,  and  infectiousness,   183 
virulent,  sublethal  doses  of,  in  active 

immunization,  195 
Bacteriaceae,   37 
Bacterial  cell,  ash  in,  23 
Babes-Ernst  granules  in,  11 
capsule  of,  12 
carbohydrates  in,  23 
chemical  constituents  of,  21 

quantitative  analysis  of,  21 

varieties  of,  21 
fats  in,  22 
globulin  in,  22 
membrane  of,  12 
metachromatic  granules  in,  11 
morphology  of,  100 
motility  of,  13 

Brownian,  14 

by  flagella,  14 


Bacterial    cell,    motility    of,    effect    of 
temperature  on,  15 
molecular,  14 
true,  14 
nucleus  in,  10 
organs  of  locomotion  of,  13 

classification  of  bacteria  based  on, 
15 
osmotic  properties  of,  23 
permeability  of  membrane  of,  23 
plasmolysis  of,  23 
plasmoptysis  of,  24 
polar  bodies  in,  11 
proteids  in,  22 

refractive  index  of  parts  of,  24 
specific  gravity  of  forms  of,  24 
water  in,  21 
Bacterial  enzymes  or  ferments,  42 
action  of,  42 

environmental  conditions  on,  43 
reversible,  43 
similarity  of,  to  ferments  of  special- 
ized cells  of  higher  plants  and  ani- 
mals, 43 
Bacterial  filtrates  for  precipitin  tests, 

preparation  of,  254 
Bacterial  forms,  variations  of,  19 
Bacterial  mutation,  38,  39 
Bacterial  poisons,  184 
action  of,  187 
ptomains  and,  185 
resistance  of,  to  chemical  action  and 

heat,  187 
summary  of,  305 
varieties  of,  185 
endotoxins,  185 
proteins,  186 
true  toxins,  185 
Bacterial  products  in  active  immuniza- 
tion, 195 
Bacterial  proteins,  186 
Bacterial  spores,  15 
formation  of,  15 
germination  of,  17     . 
position  of,  17 
varieties  of,  15 
arthrospores,  16 
true,  or  endospores,  16 


INDEX  OF  SUBJECTS 


819 


Bacterial  spores,  vegetative  forms  from, 

17 
Bactericidal    action    of    blood     serum, 

224 
Bactericidal   strengths  of  common  dis- 
infectants, 85 
Bactericidal  substances  compared  with 

agglutinins,  231 
Bactericidal  tests,  257 
in  test  tube,  257 

for  typhoid  fever,  258 
technique  of,  258 
in  vivo,  255-257 
Bacteriemia,  definition  of,  184 
Bacteriological    examination    of    blood 
cultures,  178 
choice  of  media  for,  179 
results  of,  estimation  of,  180 
technique    of    obtaining    material 
for,  178 
from  typhoid  patients,  180 
of  exudates,  175 
of  feces,  176 

of  material  from  patients,  174 
technique  of  collecting,  174 
of  urine,  176 
of  water,  693 
Bacteriological  examination  of  oysters, 

718 
Bacteriology,  development  and  scope  of, 

1-8 
Bacteriolysins,  200 

immune,  225 
Bacteriolytic  tests,  255 
Pfeiffer's  test  in,  255 

determination       of       bacteriolytic 
power  of  serum  against  a  known 
microorganism  in  vivo  by,  255 
identification  of  microorganism  in 
known  immune  serum  in  vivo  by, 
257 
Bacteriotropins,  282,  316 
Bacterium,  37 
Bacterium  termo,  455 
Bacterium  tularense,  562 
Bail,  agressin  theory  of,  292 

opposition  to,  293 
Balantidium  coli,  792 


Barsiekow's  medium  for  colon-typhoid 

diflferentiation,  138 
Bauer's    modification    of    Wassermann 

test  for  syphilis,  268 
"Bazillenemulsion,"  492 
Beggiatoa,  genus,  38 
Beggiatoacese,  38 
Berkefeld  filter,  120,  121 
Bile  medium  for  colon-typhoid  differen- 

tijjtion,  137 
Bile-salt  agar,  MacConkey's,  for  colon- 
typhoid  difl'erentiation,  138 
Bitter  milk,  bacteria  causing,  703 
Black  Death,  554 
Black  water   fever,  following   malaria, 

786 
Bladder  diseases  due  to  colon  bacillus, 

395 
"Blastocystitis  hominis,"  739 
Blastomycetes.     See  Yeasts  and  Yeast 

cells 
Blood,  laked,  225 

the  seat  of  immunizing  agencies,  198 
Blood  corpuscles,  red,  in  Ehrlich's  the- 
ory of  lytic  process  in  blood  serum, 
227 
Blood  cultures,  anaerobic,  180 

bacteriological  examination  of,  178 

results  of,  estimation  of,  180 
choice  of  media  for,  179 
technique  of  obtaining  material  for, 
178 
from  typhoid  patients,  180 
Blood  examinations  for  protozoa,  tech- 
nique of,  795 

MacNeal's  stain,  797 
Manson's  stain,  798 
Wright's  stain,  796 
Blood  media,  method  of  obtaining,  140 
Blood  serum,  bactericidal  action  of,  224 
Bordet's  interpretation  of  lytic  proc- 
esses of,  225 
immune,  198 

reactivation  of  bactericidal  power 

of,  by  normal  serum,  225 
reactivation  of  bacteriolytic  powers 
of,  by  normal  serum,  226 
lytic  processes  of,  224 


820 


INDEX  OF  SUBJECTS 


Blood  serum,  normal,  198 

Bordet's   lytic  theory  of  constitu- 
ents of,  225 
alexin  in,  225 

"sensitizing  substance"  in,  225 
obtaining  of,  from  animals,  250 

from  man,  250 
reactions  with.     See  Serum  reactions 
Blood  serum  reactions,  method  of  ob- 
taining, 139 
Bodonidae,  740 
Bollinger,  discovery  of  actinomyces  of 

cattle  by,  622 
Bordet      and      Gengou,    discovery      of 

whooping-cough  bacillus  by,  543 
Bordet-Gengou  bacillus,  543 
cultivation  of,  544 

compared    with   that    of    influenza 

bacillus,  544 
technique  of,  544-545 
morphology  of,  543 

compared    with    that   of    influenza 
bacillus,  544 
pathogenicity  of,  545 
staining  of,  543 
toxins  of,  545 
Botulism,  477 

Bouillon,    malachite    green,    for    colon- 
typhoid  differentiation,  137 
Bouillon  filtre   (Denys),492 
Bovine    tuberculosis,    bacillus    of.     See 

Tubercle  bacillus,  bacilli  related  to 
Brilliant  green  agar  for  typhoid  isola- 
tion, 136 
Broth  used  for  culture  media,  124 
calcium  carbonate,  126 
glycerin,  125 
meat  extract,  124 
meat  infusion,   124 
nitrate,  126 
pepton-salt,  126 
sugar-free,  125 
Uschinsky's  proteid-free,  126 
Bruce,  discovery  of  Malta  fever  bacil- 
lus by,  550 
Buboes,  548 

Buchner,    discovery    of    Bacillus    coli 
communis  by,  389 


Buchner's  method  of  pyrogallic  absorp 
tion    of   oxygen    in    cultivation    of 
anaerobic  bacteria,  152 
Wright's  modification  of,  153 
Buerger's  method  of  staining  capsules, 

99 
Butter  bacillus,  502 
Butter,  making  of,  710 
bacteria  aiding,  711 
transmission  of  infection  by,  711 
tubercle  bacilli  in,  711 
Butyric-acid  fermentation  in  milk,  702 

Cadaveein,  45 

Cages  for  animals,  171,  172,  173 

Calcium-carbonate  broth,  126 

Capsule  stains  in  staining  of  bacteria, 

98 
Carbohydrates  in  bacterial  cell,  23 
Carbolic  acid  as  disinfectant,  77 
Carbolic-acid  coefficient,  80 
Carbon  dioxid  formed  by  bacteria,  164 
Carbon  in  nutrition  of  bacteria,  25 
Casein,  coagulation  of,  in  milk,  702 
Castelli,   discovery   of   Spirochaeta  per- 

tenuis  by,  614 
Cell-receptors,  three  forms  of,  in  expla- 
nation of  all  known  antibodies 
(Ehrlich)  : 
haptines  of. the  first  order,  240 
haptines  of  the  second  order,  240 
haptines  of  the  third  order,  240 
Cellulase,  49 

Ceratophyllus  fasciatus,  744 
Cercomonadidse,  740 
Cercomonas  hominis,  740 
Charbon.     See  Anthrax 
Charbon  symptomatique.     See  Anthrax 

symptomatic,  bacillus  of 
Cheese,  making  of,  714 
bacteria  aiding,  714 

pathogenic  organisms  in,  714 
Chemotaxis,  negative  and  positive,  defi- 
nition of,  277 

non-specific,  291 
Chicken  cholera  bacillus,  552 

cultivation  of,  552 

immunization  with,  553 


INDEX  OF  SUBJECTS 


821 


Chicken    cholera    bacillus,    morphology 
and  staining  of,  552 
occurrence  of,  in  animals,  552 
pathogenicity  of,  552 
Chicken-pox,  relation   of,  to   smallpox, 

660 
Chlamydobacteriaceae,  37,  618 
classification  of,  618 
morphology  of  various  forms  of,  618 
Chlamydozoa,  793 

Chloride  of  lime  as  disinfectant,  76 
Chlorine  as  disinfectant,  88 
Cholera,  Asiatic,  582 
diagnosis  of,  584 
epidemics  of,  586 
immunization  in,  590 
active,  590 

protective  inoculation  in,  590 
in  animals,  587 
infection  in,  587 
pathological  findings  in,  587 
spirillum  of,  582 

bi/)logical  considerations  of,  586 

cultivation  of,  583 

diagnosis    of,    by    "cholera-red" 

reaction,  584 
Dieudonne's  selective  medium  for 

cultivation  of,  584 
Dieudonne's  selective  medium  for 
cultivation     of,     modified     by 
Krumwiede  and  Pratt,  584 
hygienic  considerations  of,  588 
in  feces,  177 
isolation  of,  585 
from  feces,  585 
from  water,  586,  694 
morphology  of,  582 
pathogenicity  of,  586 
in  animals,  587 
in  man,  587 
resistance  of,  586 
spirilla  resembling,  590 
Spirillum  Deneke;  591 
Spirillum  Massaua,  591 
Spirillum  Metchnikovi,  590 
Spirillum      of      Finkler-Prior, 
591 
staining  of,  583 


Cholera,  Asiatic,  spirillum  of,  toxin  of, 
589 
traced  to  milk,  705 
Cholera,  fowl,  bacillus  of,  7 
Cholera   infantum   attributed  to   colon 

bacillus,  394 
Cholera    nostras    attributed    to    colon 

bacillus,  394 
Cholera-red  reaction,  584 
Chromo-bacteria,  59 
Chromogenic  Gram-negative  cccci,  387 
Ciliata.     /S^e  Infusoria 
Cladothrix,  37,  619 

morphology  of,  618 
Clearing  of  culture  media,  119 
by  filtering,  120 
with  eggs,  119 
Clostridium  Pasteurianum,  54 
Coagulins,  235 

Cobra  poison  and  its  antitoxin,  experi- 
mentation with,  204 
Coccaceae,  36 
Cocci,  description  of,  9 
Coccidia,  differentiated  from  plasmodia, 

766 
Coefficient  of  inhibition,  80 
Colon  bacillus.     See  Bacillus  coli  com- 
munis 
Colon  bacillus  group,  389 
Colon  test,  for  analysis  of  water,  696 
Colon-typhoid     differentiation,     media, 
for,  132 
Barsiekow's,  138 
bile,  137 

brilliant  green  agar,  136 
Conradi-Drigalski,  134 
Endo's,   134 
Hesse's,  133 
Hiss'  plating,  133 
Hiss'  tube,  133 
Jackson's  lactose-bile,  137 
Kendall's  modification  of  Endo's,  1  Ho 
MacConkey's  bile-salt,  138 
malachite  green  bouillon,  137 
neutral-red,  138 

Russell's  double  sugar  agar.  13S 
Colon-typhoid-dysentery    group,    bacilli 
of,  388 


822 


INDEX  OF  SUBJECTS 


Colon-typhoid-dysentery    group,    bacilli 

of,  characteristics  of,  388 
Colonies  in  agar,  146 
Colony-fishing,  146 
Colony  study  of  bacteria,  161 
Color  indicator  in  titration,  117 
Comma  bacillus.    See  Spirillum  cholerse 

asiaticae 
Complement,  deviation  of,  246 
division  of,  into  fractions,  245 
filtration  of,  244 

fixation  of.     See  Complement  fixation 
further  facts  and  theories  concerning, 

242-245 
in   Ehrlich's  theory  of   lytic  process 

in  blood  serum,  226 
in  normal  blood  serum,  226 
inhibition  of,  245 

multiplicity  of,  in  normal  sera,  243 
quantitative  relationship  between  am- 
boceptor and,  246 
Complement  fixation,  action  in,  246 
by  precipitates,  245 
determination  of  antigen  by,  in  serum 
reactions,  271 
of  antibodies  in  sera  by,  261 
principles  of,  261 
reaction  in,  261 

Wassermann    test   for.     See    Was- 
sermann    test    for    diagnosis    of 
syphilis 
in  glanders,  535 
in  tuberculosis,  494 
proteid  differentiation  by,  273 
Complementoids,  244 
Conjunctivite  subaigue,  545 
Conradi-Drigalski    medium,    for    colon- 
typhoid  differentiation,  134 
isolation  of  typhoid  bacillus  in  stools 
by,  408 
Cotton  used  in  filtering  culture  media, 

120 
Counting  of  bacteria,  161 
Cowpox,  relation  of,  to  sn\allpox.  659 
use     of,     in     immunization     against 
smallpox,  659 
Crenothrix,  37 
Creolin,  78 


Cultivation  of  bacteria,  anaerobic,  148 
by  absorption  of  oxygen  by  pyro- 
gallic  acid  in  alkaline  solu- 
tions,  152 
Buchner's  method,  152 

Wright's     modification     of, 
153 
combined    with    air    exhaustion 
and  hydrogen  replacement,  153 
on  agar  slants,  153 
without  use  of  hydrogen,  155 
by  displacement  of  air  with  hydro- 
gen, 151 
by  mechanical  exclusion  of  air,  148 
Esmarch's  method,  149 
fluid  media  covered  with  oil,  150 
Liborius'  method,  149 
Roux's  method,   149 
Wright's  method,  150 
Zinsser's  apparatus  for,  156 
colony  study  in,  161 
counting  of  bacteria  in,  161 
incubation  in,  158 
See  also  Incubation  of  cultures 
Culture  media,  124 
agar  for,  127 

lactose-litmus,   129  ' 

meat  extract,  127,  135 
meat  infusion,  128 
blood  for,  140 
blood  serum  for,  139 
broth  for,   124 

calcium  carbonate,  126 
glycerin,  125 
meat  extract,  124 
meat  infusion,  124 
nitrate,   126 
pepton-salt,  126 
sugar-free,  125 
Uschinsky's  proteid-free,  126 
clearing  of,  119 

by  filtering  through  cotton,  120 
through"  paper,  121 
with  eggs,  119 
Dorsett  egg,  130 

enriching  substances  used  in,  138,  139 
fiuid,  covered  with  oil,  in  anaerobic 
cultivation  of  bacteria,  150 


INDEX  OF  SUBJECTS 


823 


Culture    media,    for   colon-typhoid    dif- 
ferentiation, 132 
glassware  preparation  in,  113 
glycerin  for,  126 
ingredients  of,  115 

choice  of,  116 
lactose  litmus-agar,  129,  134 
litmus  milk,  130 
milk,  130 
potato  for,  130 
glycerin,   130 
preparation  of,  113 

general  technique  of,  113 
process  of,  124-140 
reaction  of,  119 
serum,  131 

Hiss'   serum   water,  for   fermenta- 
tion tests,  132 
Loeffler's,   131 
slanting  of,  123 

special,  132 
sterilization  of,  121 
filtration  in,   122 
heat  in,  121 
titration  of,  117 

color  indicator  in,  117 
process  of,  117 

for  alkaline  media,  118 
reaction  of,  117 
adjustment  of,  119 
tubing  of,  121 
Cultures,   attenuated,   in  active   immu- 
nization, 193 
Cultures,   incubation   of.      See   Incuba- 
tion of  cultures 
Cytase,   279 
Cytoryctes  variolsB,  658 
Cytotoxins,  201 

specific,  injury  to  organs  by,  201 

Dark-field  condenser,  597 

Decay,  action  of,  44 

Defensive  factors  of  animal  organism, 

189 
Degenerative  forms  of  bacteria,  20 
Delhi  coil,  organism  of,  758 
Denitrifying  bacteria,  52 

activities  of,  53 


Denitrifying    bacteria,    occurrence    of, 

53 
"Derrengaderas  de  caderas,"  747 
Destruction  of  bacteria,  62 
by  chemical  agents,  73 
See  also  Disinfectants 
gaseous,  88 
in  solution,  73 
inorganic,  73 
organic,  77 
by  physical  agents,  62 
drying,  62 
electricity,   65 
heat,  65 
light,  63 
Diarrheal  diseases  traced  to  milk,  705 
Diastase.     See  Amylase 
Differential  stains  in  staining  of  bac- 
teria, 102 
Diphtheria,  tracing  of,  to  milk,  705 
Diphtheria  antitoxin,  216 

anaphylaxis  in  injections  of,  296 
determination     of     presence     of,     in 

blood,  by  Shick  reaction,  526 
normal,  205 
production  of,  216 

horses  used  in,  216-217 
individual,    by   injection  of  toxin- 
antitoxin  mixture,  527 
technique  of,  217 
toxin  for,  216 
stable,  206 
standardization  of,  218 

concentration  and  purifying  in,  219 
technique  of,  218 
unit  for,  205 
Diphtheria  bacillus,  512 
bacilli  similar  to,  523 
Bacillus  Hoffmanni,  523 
cultivation  of,  524 
morphology  of,  523 
staining  of,  524 
Bacillus  xerosis,  524 
cultivation  of,  525 
differentiation  of,   from  other  ba- 
cilli, 525 
other  bacilli,  525 
biological  characteristics  of,  616 


824 


INDEX  OF  SUBJECTS 


Diphtheria  bacillus,  cultivation  of,  516 
degenerative  forms  of,  19 
determination  of  virulence  of,  519 
differentiation  of,  from  other  forms, 

107,  523  et  seq. 
grouping  of,  515 
isolation  of,  517 
morphology  of,  513 

"ground"  type  in,  514 
occurrence  of,  in  body,  518,  519 
pathogenicity  of,  518 
in  animals,  519 
**pseudo-membranes"  in,  518 
resistance  of,  515 
staining  of,  574 

Gram's  method  of,  507 
polar  or  Babes-Ernst  bodies  in,  514 
Neisser's  stain  for,  514 
Roux's  stain  for,  515 
toxin  of,   520.     See  also  Diphtheria 
toxin 
chemical    and    physical    properties 

of,  521 
production  of,  520 

media  employed  in,  520 
Park-Williams  Bacillus  No.   8  in, 

520 
resistance  of,  522 
Diphtheria  carriers,  522 
Diphtheria    toxin,    analysis    of     (Ehr- 
lich),  205-215 
method    of   partial   absorption    in, 

209 
side-chain  theory  of,  212 
summary  of,  215 
constitution  of   (Ehrlich),  210. 
graphic  form  of   (Ehrlich),  211 
views  of  Arrhenius  and  Madsen  on, 
212 
epitoxoid  in,  208 
molecule  of,  haptophore  group  in,  207 

toxophore  group  in,  207 
neutralized,       active      immunization 

with,  527 
normal  solution  of,  205 
partial  absorption  of,  209 
standardization  of.   Limes  death  in, 
208 


Diphtheria    toxin,    standardization    of. 
Limes  zero  in,  2:07 
time  changes  in,  206 
toxoid  form  of,  207 
protoxoids  in,  209 
syntoxoids  in,  209 
toxon  in,  209 
unit  for,  205 
Diphtheroid  bacilli,  526 
Diplococcus  gonorrhoeae,  380 
antibodies  to,  385 
cultivation  of,  381 

conditions  favorable  to,  383 
Wertheim's  medium  for,  381-382?: 
differentiation   of,   from   Micrococcus 

catarrhalis,   386 
early  work  in,  380 
infection  by,  in  man,  384 
morphology  of,  380 
Pappenheim-Saathof  stain  of,  381 
pathogenicity  of,  in  animals,  384 

in  man,  384 
resistance  of,  383 
staining  of,  381 
Diplococcus  lanceolatus.     See  Diplococ- 
cus pneumoniae 
Diplococcus  mucosus,  387 
Diplococcus  pneumoniae,  352 
cultivation  of,  355 
differentiation  of,  from  streptococcus, 
357,  367 
cultural,  369 
morphological,  367 
by  bile  test,  370 
by  capsule,  368 
by  fermentations,  369 
by  grouping,  367 
by  growth  in  blood  media,  367, 

370 
by  shape,  367 
immunization  against,  362 
active,  methods  of,  363 
agglutinins  in  immune  sera  in,  363 
table  of,  364 
technique  of,  366 
precipitins  in  immune  sera  in,  365 
passive,  with  immune  sera,  365 
isolation  of,  358 


INDEX  OF  SUBJECTS 


825 


Diplococcus   pneumoniae,   modes   of    in- 
oculation with,  3G1 
morphology  of,  553 
capsules  in,  353-354 
lancet-shape  of  cocci  in,  353 
pairing  of  cocci  in,  353 
pathogenicity  of,  359 
pneumonic  complications  and,  362 
resistance  of,  358 
staining  of,  355 

susceptibility  of  animals  to,  370 
toxic  products  of,  362 
^treatment  of,  367 
virulence  of,  359 
in  animals,  359 
in  man,  361 
Disinfectants,  73 

bactericidal  strengths  of  common,  85 
gaseous,  88 
chlorine,  88 
formaldehyde,  89 
oxygen,  88 
ozone,  88 

sulphur  dioxide,  88 
testing  of  efficiency  of,  79 

by    U.    S.    Public    Health    Service 

method,  80 
carbolic-acid  coefficient  in,  80 
determination  of  antiseptic  values 
in,  83 
of  disinfectant  values  in,  86 
table  of,  85 
factors  in,  79 
used  in  solution,  73 
inorganic,  73 

acids,  bases,  and  salts,  74 
halogens  and  derivatives,  75 
chlorid  of  lime,  76 
tetrachlorid  of  iodin,  76 
hydrogen  peroxid,  76 
potassium  permanganate,  76 
organic,  77 
alcohols,  77 
carbolic  acid,  77 
cresol  group,  78 
essential  oils,  78 
formaldehyde,  78 
iodoform.  77 


Disinfection,  practical,  86 
of  feces,  87 
of  hands,  87 
of  instruments,  87 
of  linen,  etc.,  87 
of  rooms,  etc.,  88 
of  sputum,  87  « 

of  urine,   87 
Dissociation,  electrolytic,  74 

relation    between    degree    of,    and 
bactericidal  powers  of  solutions, 
74 
Dor  sett  egg  medium,  130 
Dourine,  chemical  course  of,  748 

diagnosis  of,  by  complement-fixation, 

749 
transmission  of,  748 
Drying  in   destruction  of  bacteria,  re- 
sistance to,  62 
DTNiM25,o,  definition  of,  205 
Ducrey  bacillus,  547 

cultivation  and  isolation  of,  548 
discovery  of,  548 
infection  with,  547 
pathogenicity  of,  549 
Dysentery,  amebic.     See  Amebic  Dys- 
entery 
Dysentery,  autopsy  findings  in,  442 
occurrence  of,  442 
symptoms  of,  442 
Dysentery  bacilli,  435 

biological  consideration  of,  441 
differentiation   of,   by  Hiss,   through 
agglutination  tests,  440 
through  fermentation  tests,  439 
immunization  with,  444 
active,  444 

agglutinins  in,  444 
bactericidal  substances  in,  444 
true  toxins  in,  445 
passive,  445 
in  feces,  178 

investigation  of,  by  Flexner,  436 
by  Hiss  and  Russell,  438 
by  Kruse,  437 
by  Lentz,  438 
by  Martini  and  Lentz,  438 
by  Ohno,  439 


826 


INDEX  OF  SUBJECTS 


Dysentery  bacilli,  investigation  of,  by 
Park  and  Carey,  439 

by  Park  and  Dunham,  438 

by  Shiga,  435 

by  Spronck,  437 

by  Strong  and  Musgrave,  436 

by  Vedder  and  Duval,  437 
pathogenicity  of,  441 
pseudo-dysentery   bacillus    and,   437, 

438 
Shiga's  bacillus  in,  435 

cultural  characteristics  of,  436 

morphology  of,  435 
toxic  products  of,  442 

action  of,  on  animals,  443 

obtaining  of,  from  cultures,  442 
"Y"  bacillus  in,  438 

Ebebth,  discovery  of  typhoid  bacillus 

by,  399 
Edema,    malignant,    bacillus    of.      See 

under  Malignant  edema 
Eel-blood  serum,  toxic,  205 
Eggs  used  in  clearing  culture  media, 

119 
Ehrlich's  analysis  of  diphtheria  toxin, 

205-215.     See  also  under  Diphtheria 

toxin 
Eichstedt,    discovery    of    Microsporon 

furfur  by,  639 
Electricity  in  destruction  of  bacteria, 

65 
Electrolytic  dissociation,  74 

relation  between  degree  of  and  bac- 
tericidal powers  of  solutions,  74 
Elser  and  Huntoon,  discovery  of  pseu- 

domenigococcus  by,  379 
Eisner's    potato-extract    gelatin,    isola- 
tion of  typhoid  bacillus  in  stools  by, 

407 
Emphysematous  gangrene,  isolation  of 

Bacillus   aerogenes   capsulatus  from, 
474 
Endameba,  723 

forms  of,  723 

Endameba  africana,  731 
Endameba  coli,  735 
morphology  of,  734 


Endameba,    forms    of,    Endameba    coli, 
multiplication  of,   735 
nature  and  occurrence  of,  734 
pathogenicity  of,  736 
size  of,  734 
Endameba  gingivalis,  736 
cyst  formation  of,  734 
morphology  of,  736 
occurrence  of,  736 
pathogenicity  of,  736 
site  of,  736 
Endameba  histolytica,  725 
as  cause  of  dysentery,  726 
cyst  formation  of,  731 
degenerative  forms  of,  731 
morphology  of,  729 
pathogenicity  of,  726 
Endameba  tetragena,  731 
pathogenic  history  of,  725 
Endolysins,  291 

Endo's    fuchsin-agar,    isolation    of    ty- 
phoid bacillus  in  stools  by,  409 
Endo's  medium,  for  colon-typhoid  dif- 
ferentiation, 134 
Harding  and   Ostenberg's  method  of 

preparation  of,  135 
Kendall's  modification  of,  135 
Endospores,  16 
Endotoxins,  185,  306 

compared  with  pigments,  186,  309 
of    Staphylococcus   pyogenes   aureus, 

328 
of  Streptococcus  pyogenes,  345 
summary  of,  306 
toxins  distinguished  from,  186 
Environment  and  bacteria,  31 
Enzymes,  definition  of,  42 
katalyzers  and,  analogy  between,  42 
produced  by  bacteria,  168 
diastatic,  169 
inverting,  169 
proteolytic,  168 
varieties     of.      See     under     specific 
names 
Epitoxoid  in  toxin,  208 
Erlenmeyer  flask,  114 
Escherich,  discovery  of  Bacillus  lactis 
aerogenes  by,  453 


INDEX  OF  SUBJECTS 


827 


Esmarch  roll  tubes  in  isolation  of  bac- 
teria,  147 
Esmarch's  method  of  anaerobic  cultiva- 
tion of  bacteria,  149 
Essential  oils  as  disinfectants,  78 
Eumycetes.     See  Hyphomycetes 
Exudates,    bacteriological    examination 
of.     See  Bacteriological  examination 


Facultative  aerobes,  26 
Facultative  anaerobes,   26 
Farcy,  531 
Fat-splitting  enzymes,  47 

action    of,  method   of    investigating, 

47 
varieties  of,  47 
Fats  in  bacterial  cell,  22 
Favus,  642 

Feces,    bacteriological    examination   of, 
176 
disinfection  of,  87 
number  of  bacteria  in,  176 
varieties  of  bacteria  in,  177 
Fermentation,  alcoholic,  51 
in  milk,  702 
butyric-acid,  in  milk,  702 
enzymes  of,  48,  52 
in  development  of  bacteria,  26 
inversion  in,  42 
lactic-acid,  50,  701 
process  of,  48 

tests,  serum  media  for,  132 
Filtering,  in  clearing  of  culture  media, 
102 
through  cotton,  120 
through  paper,  121 
in  sterilization  of  culture  media,  122 
Filters,  varieties  of: 
Berkefeld,  120,  121 
Kitasato,  123 
Maassen,  124 
Reichel,  122 
Filtrable   table   compiled   by  Wolbach, 

682 
Filtrable  virus,  681 

Filtration  of  immune  body  and  comple- 
ment, 245 


Fishing,  colony,  146 

Fixation  of  complement,  action  in,  247 

by  precipitates,  245 
"Fixator,"  279 
Flagella,  arrangement  of,  15 
structure  of,  14 
varieties  of,   14 
Flagella  stains  in  staining  of  bacteria, 

100 
Flagellata.    See  Mastigophora 
Florence  flask,  114 
Fluid  media  covered  with  oil,  used  in 

cultivation  of  bacteria,  150 
Foot-and-mouth  disease,  680 
etiological  factor  of,  681 
immunity  in,  681 
in  milk,  706  , 

pathology  of,  680 
transmission  of,  680 
Formaldehyde  as  disinfectant,  78,  89 
generation  of,  by  breaking  up  solid 
polymer  with  heat,  91 
by  Breslau  method,  90 
by  direct  evaporation,  89 
by  glycerine  addition,  90 
by  lime  method,  91 
by  potassium-permanganate  meth- 
od, 92 
by  Trillat  method,  89 
gauging  amount  of  formalin  in,  92 
Trillat  autoclave  for,  89 
Fractional  sterilization,  70 
at  high  temperatures,  70 
at  low  temperatures,  71 
Framboesia  tropica,  614 
Friedlander,  discovery  of  Bacillus  mu- 

cosus  capsulatus  by,  447 
Friedlander  bacillus.    See  Bacillus  mu- 

cosus  capsulatus 
v.  Frisch,  discovery  of  Bacillus  of  rhi- 

noscleroma,  451 
Fumigation.    See  Disinfectants,  Disin- 
fection 
Fusiform  bacilli,  614 

from  carious  teeth,  613 
•  from  noma,  614 
from  scurvy,  614 
of  Vincent's  angina,  612 


828 


INDEX  OF  SUBJECTS 


Gaffky,  discovery  of  Micrococcus  tetra- 

genus  by,  334 
Gartner,   discovery  of  Bacillus   enteri- 

tidis  by,  429 
Gas  bacillus  infections,   in  soldiers  in 

trenches,  474 
Gas  formed  by  bacteria    164 
analysis  of,  164 

carbon  dioxid  in,  164 
hydrogen  in,  165 
hydrogen  sulphid  in,  165 
quantitative,  166 
Gas-pressure  regulators  for  incubators, 
160 
Moitessier's,  160 
Gastrointestinal  autointoxication,  715 
bacteria  causing,  715 
experimental  combating  of,  by  acid- 
producing  bacilli,  716 
Metchnikoff's  therapy  of,  by  means  of 
Bacillus  bulgaricus,  716-717 
Gelase,  49 
Gessard,   discovery   of   Bacillus   pyocy- 

aneus  by,  578 
Glanders,   bacteriological   diagnosis   of, 
532 
complement  fixation  in,  535 
irjimunity  in,  535 
in  horses,  530 
acute  form,  530 
chronic  form,  531 
in  man,  532 
nodules  of,  532 
spontaneous  infection  in,  530 
Glanders   bacillus.     See   Bacillus   mal- 
lei 
Globulin  in  bacterial  cell,  22 
Glossina  morsitans,  754 
Glossina  palpulis,  752 
Glycerin,  use  of,  for  culture  media,  126 
meat  extract,  126 
meat  infusion,  127 
Glycerin  broth,  125 
Glycerin  potato,   130 
Gonococcus.     See     Diplococcus     gonor- 
rhoeae 
Gram-negative  bacteria,  104 
Gram-negative  cocci,  chromogenic,  387 


Gram-negative  micrococci,  table  of  di- 
agnosis of,  by  diflFerential  value  of 
sugar  fermentation,  387 

Gram-positive  bacteria,  104 
in  feces,  177 

method  of  staining  in  tissue  sections, 
111 

Gram-Weigert  method  of  staining 
gram-positive  bacteria  in  tissue  sec- 
tions, 111 

Gram's  method  of  staining  bacteria,  102 
classification   of   pathogenic  bacteria 

by,  104 
Paltauf's  modification  of,  103 
Sterling's  modification  of,  103 

Group  agglutination,  234 

Gruber-Widal  reaction,  251 

Gruby,  discovery  of  Trichophyton  ton- 
surans by,  643 

Guarnieri's  medium,  Welch's  modifica- 
tion of,   129 


Haematopinus  Spinulosus,  744 
Halogens  as  disinfectants,  75 
Halteridium.     See  Hemoproteus  colum- 

bse 
''Hanging  block"   method   in   study    of 

bacteria,  94 
"Hanging   drop"    method    in    study    of 

bacteria,  93 
Hansen,  discovery  of  lepra  bacillus  by, 

505 
Haptophore   group   in   toxin   molecule, 
207 
in  toxon  molecule,  209 
Haptophore   groups    in    immune   body, 
228 
complementophile,  228 
cytophile,  228 
Hauser,   discovery   of  Bacillus  proteus 

vulgaris  by,  454 
Heat,  in  destruction  of  bacteria,  65 
dry  and  moist,  comparison  of,  66 
effect  of  various  degrees  of,  65 
moist,  advantages  of,  66 
penetrating  power  of,  67 
in  sterilization  of  culture  media,  121 


INDEX  OF  SUBJECTS 


82f^ 


Heat  sterilization,  68 
dry,  68 

by  burning,  68 
by  hot  air,  68 
moist,  69 

by  boiling,  69 

by  fractional  sterilization,  70 
by  live  steam,  60 
by  steam  under  pressure,  71 
Hektoen  and  Ruediger  on  structure  of 

opsonins,  283 
Hemagglutinins,  235 
Hematogen,  538 
Hemolysin,  immune,  226 
Hemolysins  of  Staphylococcus  pyogenes 
aureus,  329 
of  Streptococcus  pyogenes,  344 
specificity  of,  248 
varieties  of: 
autolysins,  248 
heterolysins,  248 
isolysins,  248 
Hemolysis,  201,  225 
Hemolytic  serum,  265 
Hemolytic  tests,  259 

obtaining  blood  for,  259 
in  large  quantities,  260 
in  small  quantities,  259 
standard  concentration  of  red  blood 
cells  for,  259 
Hemolytic  unit,  definition  of,  265 
Hemoproteus  columbae,  form  and  devel- 
opment of,  761 
invertebrate  host  of,  761 
life  history  of,  763 
occurrence  of,  761 
Hemoproteus  noctuae,  750 
Hemoproteus  ziemani,  751 
Hemorrhagic  septicemia  bacilli,  551 
morphology  of,  551 
staining  of,  551 
varieties  of: 

bacillus  of  chicken  cholera,  552 
Bacillus  pestis,  554 
bacillus  of  swine  plague,  553 
Hemosporidia,    form    and    development 
of,  760       . 
hemoproteus  columbse.  76 1 


Hemosporidia,  pinoplasmidse,  787 
proteosoma     (plasmodium)     praecox, 
763 

Hepatotoxin,  201 

Herpetomonada,  755 

Hesse's   medium  for  colon-typhoid  dif- 
ferentiation, 133 

Heterolysins,  248 

Hides,  tanning  of,  bacteria  in,  720 

Hiss'  agar-gelatin  medium,  isolation  of 
typhoid  bacillus  in  stools  by,  407 

Hiss'     leucocyte     extract    theory     and 
therapy,  289 

Hiss'  methods  of  staining  capsules,  98 

Hiss'   plating  media  for  colon-typhoid 
differentiation,    133 

Hiss'    tube    medium    for    colon-typhoid 
differentiation,   133 

Hiss'  serum  water  media  for  fermenta- 
tion tests,  132 

"Hog-cholera"  bacilli,  430 

differentiation  of,  from  swine  plague 
bacillus,  554 

Hogyes,   dilution  method  of,   in  treat- 
ment of  rabies,  656 

Horse  meat,  detection  of,  by  precipitin 
tests,  254 

Horses    used    in    production    of    diph- 
theria antitoxin,  216-217 
of  tetanus  antitoxin,  221 

Howard     and     Perkins,     discovery     of 
Streptococcus  mucosus  by,  350 

Huntson's  method  of  staining  capsules, 
98 

Hydrocyanic    gas,    destruction    of    ro- 
dents by  fumigation  with,  92 

Hydrogen,    formation    of,    by   bacteria, 
165 
in  nutrition  of  bacteria,  28 

Hydrogen  peroxid  as  disinfectant,  76 

Hydrogen  sulphid  formed  by  bacteria, 
165 

Hydrophobia.     *Sfee  Rabies 

Hypersusceptibility.      See   Anaphylaxis 

Hyphomycetes,   635 

conditions    favorable    to    growth    of, 
■  638 
diseases  caused  by,  639 


830 


INDEX  OF  SUBJECTS 


Hyphomycetes,  diseases  caused  by,  fa- 
vus,  642 
pityriasis  versicolor,  639 
ringworm,  643 
thrush,  640 
diseases  sometimes  accompanied  by, 

644 
monilia,  641 
morphology  of,  635 
reproduction  of,  635 
mycomycetes  in,  636 

Aepergillus  variety  in,  637 
Penicillium  variety  in,  637 
sporulation  by  other  methods  in, 
637 
phycoraycetes    in,    Mucorinae    vari- 
ety of,  635 
sexual  reproduction  of,  636 
structural  classification  of,  635 
varieties  of,  635 
mycomycetes,  635 

morphology    of     (typical),    635, 
636 
ascospores  in,  638 
conidia    (or  spores)    in,  638 
conidiophores  in,  637 
hyphal  branches    (septate)   in, 

637 
mycelial  threads  (septate)   in, 

637 
sterigmata   (septate)    in,  637 
morphology     of     (less    frequent 
forms  of),  637 
ascospores  in,  637 
chlamydospores  in,  638 
phycomycetes,  635 

morphology    of    (typical),    635- 
636 
columella  in,  636 
hyphal  branches  in,  636 
mycelial  threads  (non-septate) 

in,  636 
sporangium  in,  636 
spores  in,  636 
morphology  of  sexual  reproduc- 
tion forms  of,  636 
gametophores  in,  636 
zygospores  in,  636 


"Immune  body'*  in  blood  serum,  226 
in  Ehrlich's  theory  of  lytic  process  in 
blood  serum,  226 
haptophore  groups  of,  228 
complementophile,  228 
cytophile^  228 
Immunity,  absolute,  190 
acquired,  192 

definition  of,  190,  192 
active,   193  '  , 

artificial,  193 
definition  of,   193 
experimentation    with     attenuated 
cultures  for,  193 
with  bacterial  products  for,  195 
with  dead  bacteria  for,  195 
with  sublethal  doses  of  fully  vir- 
ulent bacteria  for,  195 
definition  of,  189 
natural,   190 
individual,  191 
of  races,  191 
of  species,  190 
passive,  196 

relation   of,-  to   phagocytotic  powers 
in  animals,  279 
Immunization,  active,  with  neutralized 
diphtheria  toxin,  527 
blood  the  seat  of,  198 
Incubation  of  cultures,  156 
incubators  in,  158 
gas-pressure  regulators  for,  160 
thermo-regulators  for,  159 
Indigo  production,  bacteria  in,  720 
Individual  immunity,  191 
in  higher  animals,   192 
in   lower    animals,    191 
Indol  production  by  bacteria,  167 
Industries,  bacteria  in,  719 
Infantile    splenomegaly,    organism   of, 

758 
Infection,  definition  of,  181 
fundamental  factors  in,  181 
paths  of,   183 
inner,   184 
outer,  183 
Infectiousness,  definition  of,  182 
due  to  number  of  bacteria,  182 


INDEX  OF  SUBJECTS 


831 


Infectiousness,    due    to    variations    in 

virulence,  183 
Influenza,  epidemic  of,  in  1889-90,  536 

organs  attacked  in,   540 
Influenza  bacillus,  536 
bacteria  related  to,  541 
Bacillus  murisepticus,  542 
bacillus  of  pleuro-pneumonia,  542 
Bacillus  rhusiopathiae,  542 
Koch-Weeks  bacillus,  542 
pseudo-influenza  bacillus,  541 
biology  of,  540 

isolation  and  cultivation  of,  537 
on  agar  and  gelatin,  537 
blood  added  in,  537 
hematogen  added  in,  538 
morphology  and  staining  of,  536 
pathogenicity   of,   540 

in  experimental  inoculation  of  ani- 
mals, 541 
organs  attacked  in,  540 
susceptibility  of  animals  to,  541 
Infusoria,  balantidium  coli,  792 
chlamydozoa,  793 
classes  of,  792 

distinguishing  features  of,  792 
Inhibition,  coefficient  of,  80 
Inhibition  strengths  of  various  antisep- 
tics, 84 
Inoculation  of  animals,  170 
intraperitoneal,  172 
intravenous,  172 
subcutaneous,  171 
Inoculation  of  media,  141 

technique  of  transferring  bacteria  in, 

141 
virus   used   in  transferring  bacteria 
in,  141 
Insusceptibility     of    cold-blooded     ani- 
mals, 190 
Intravital    method'    of    Nakanishi     in 

study  of  bacteria,  94 
Inulin  media,  132,  369 
Invasion,  paths  of,  183 
inner,  184 
outer,  183 
Inversion  by  fermentation,  42 
Invertase,  49 


Iodoform  as  disinfectant,  77 
lodin,  tetrachlorid  of,  as  disinfectant, 
76 
tincture  of,  for  skin  sterilization,  76 
Iron   compounds   in   nutrition   of   bac- 
teria, 29 
Iso-agglutinins,  261 

table  for,  261 
Isohemolysins,  261 
Isolation  of  bacteria,  142 
early  methods  of,  143 
present  methods  of,  143 

Esmarch  roll  tubes  in,   147 
Koch's  plates  in,  144 
surface  streaking  in,  148 
Isolysins,  248 

Israel,  discovery  of  actinomyces  of  man 
by,  622 


Jackson's  lactose-bile  medium  for  co- 
lon-typhoid differentiation,   137 

Jenner's  discovery  of  immunization  in 
smallpox  by  vaccinia,  659 


Kala-azar,    due    to    leishmania    dono- 
vani,  757 

symptoms  of,  757 
Katalyzers,   definition   of,   42 

enzymes  and,  analogy  between,  42 
Kefyr,  702 

Kendall's  modification  of  Endo's  medi- 
um for  colon-typhoid  differentiation, 

135 
Kitasato,  discovery  of  Bacillus  tetani 
by,  456 

discovery  of  plague  bacillus,  by,  554 
Kitasato  filter,  123 
Klebs,  discovery  of  diphtheria  bacillus 

by,  512 
Koch,  discovery  of  cholera  spirillum  by, 
582 

discovery^of  tubercle  bacillus  by,  479 
Koch   plates    in   isolation   of   bacteria, 

144 
Koch- Weeks  bacillus,  542 
Koumys,   702 


832 


INDEX  OF  SUBJECTS 


L+,   definition   of,  208 
Lo,  definition  of,  207 
Lab  enzymes,  46 
Lactase,  50 

Lactic-acid  bacilli  in  therapy  of  gastro- 
intestinal autointoxication,  715 
Lactic-acid  fermentation,  50 
bacteria  of,  50 
in  milk,  701 
Lactose-bile     medium,     Jackson's,     for 

colon-typhoid  differentiation,   137 
Lactose-litmus  agar,  129 
Laked  blood,  225 
Lamblia  intestinalis,  739 
Langenbeck,   discovery  of  Oidium  albi- 
cans by,  640 
Lautenschlager's  thermo-regulator,  158, 

159 
Leishmania,  755 
Leishmania  braziliensis,  759 
Leishmania    donovani,    animal    patho- 
genicity of,  758 
as  cause  of  Kala-azar,  757 
morphology  of,  757 
transmission  of,  758 
Leishmania  infantum,  758 
Leishmania  nilotica,  759 
Leishmania  tropica,  758 
Leprolin,  502 
Leprosy,  505 

bacillus  of,  502,  505 
cultivation  of,  506 
by  Clegg,  506 
by  Duval,  506 
differentiation  of,  from  tubercle  ba- 
cillus by  staining,  106,  506 
inoculation  with,  507,  509 
morphology  of,  505 
occurrence  of,  in  body,  508 
pathogenicity  of,  507 
relation    of,    to    tubercle    bacillus, 

509 
staining  of,  505 
toxic  products  of,  509 
clinical  varieties  of,  508 
contagiousness  of,  509 
in   rats,   510 
occurrence  of,  507 


Leprosy,  tuberculin  administered  in, 
results  of,  510 

Leptothrix,  619 

morphology  of,  618 

Leucocidin  of  Staphylococcus  pyogenes 
aureus,  329 
action  of,  upon  leucocytes,  330 
discovery  of,  329 

leucotoxin  differentiated  from,  331 
obtaining  of,  330 

Leucocyte  extract,  289 

effect  of,  upon  infections  in  animals, 
290 
in  man,  291 
experimentation  with,  291 
obtaining  of,  290 

Leucotoxin,  201,  331 

Liborius'  method  of  anaerobic  cultiva- 
tion of  bacteria,   149 

Light  in  destruction  of  bacteria,  63 
action  of,  64 
resistance  to,  63 
varieties  of,  64 

Lime,  chlorid  of,  as  disinfectant,  76 

Limes  death,  definition  of,  208 

Limes  zero,  207 

"Limex  amebse,"  723 

Linen,  etc.,  disinfection  of,  87 

von  Lingelsheim,  discovery  of  Diplococ- 
cus  mucosus  by,  387 
discovery   of   Micrococcus    pharyngis 
siccus  by,  387 

Lipase,  47 

Liver  and  gall-bladder,  inflammatory 
conditions  of,  attributed  to  colon 
bacillus,  395 

Lobar  pneumonia,  infectiousness  of,  352 

Loeffier  and  Schiitz,  discovery  of  glan- 
ders bacillus  by,  528 

Loefiler's  malachite-green  media,  isola- 
tion of  typhoid  bacillus  in  stools  by, 
409 

Loefiler's  method  of  staining  flagella, 
100 

Loefiler's  serum  medium,  131 

Lustgarten,  discovery  of  smegma  ba- 
cillus by,  503 

Lyncbia  lividocolor,  761 


1 


INDEX  OF  SUBJECTS 


833 


Lynchia  maura,  761 
Lysins,  224 

action  of,  Bordet's  interpretation  of, 
225 
compared    with    Ehrlich's,    225, 

228 
summary  of,  228 
Ehrlich's  theory  of,  226 

compared  with  Bordet's,  226 
complement  in,  226 
immune  body  in,  226 
formation  of,  226 
haptophore  groups  in,  228 
complementophile,  228 
cytophile,  228 
red  corpuscles  in,  227 
side  chains  or  receptors  in,  226 
over-production  of,  226 
in  blood  serum,  225 
experimentation   in,  224 
in  immune  blood  serum,  reactivated 

by  normal  serum,  226 
in  normal  blood  serum,  Bordet's  the- 
ory of,  226 
alexin  in,  225 

sensitizing  substance  in,  225 
investigation  of,  by  Ehrlich,  226 
Lysol,  78 
Lyssa.     See  Rabies 

Maassen  filter,  124 
MacConkey's  bile-salt  agar,  for  colon- 
typhoid  differentiation,  138 
MacNeal's  stain,  797 
Macrophages,  definition  of,  276 
Madura  foot.     See  Mycetoma 
Malachite-green    media,    for    colon-ty- 
phoid    differentiation,    bouillon, 
137 
LoeflSer's,  for  isolation  of  typhoid 
bacillus  in  stools,  409 
Malaria,    black-water    fever    following, 
786 
carriers  of,  776 

€pidemiology    of,    personal     prophy- 
laxis in,  783 
precautions    against    human    car- 
riers, 783 


Malaria,    epidemiology    of,    prevention 
of  mosquito  breeding,   782 
proper  treatment   and  care  of   all 

cases,  783 
quinine  prophylaxis,  783 
screened  houses,  781 
geographical  distribution  of,  765 
history  of,  764 
immunity  to,  784 
latent,  776 
mortality  from,  766 
organism  of,  764 
pathology  of,  784 
relapses  of,  785 
treatment  of,  786 
Malarial  fever,  classical,  symptoms  of, 

775 
Malarial  fevers,  776 

clinical  description  of,  775 
incubation  period  of,  775 
Malarial  mosquito,  breeding-places  of, 
780 
classifications  of,  780 
description  of,  778 
development  of,  778 
development  of  malarial  parasite  in, 

776 
egg-laying  of,  781 
larvae  of,  778 
life  history  of,  780 
Malarial  parasite,  cultivation  of,  in  vi- 
tro, 777 
development  of,  in  the  mosquito,  776 
incubation  period  of,  781 
Malarial  parasites,  786 
Malignant  edema,  bacillus  of,  468 
cultivation  of,  470 
early  investigation  of,  469 
immunity  in,  471 
morphology  of,  469 
pathogenicity  of,  479 
staining  of,  470 
Mallein,  532 
action  of,  533 
diagnostic  use  of,  533 

directions  of  U.  S.  government  for, 
534 
obtaining  and  preparation  of,  633 


834 


INDEX  OF  SUBJECTS 


Malta  fever,  549 

in  domestic  animals,  549 
Maltase,  50 
Hanson's  stain,  798 
Maragliano's    serum    for    tuberculosis, 

498 
Marchiafava    and    Celli,    discovery    of 

meningococcus  by,  371 
Margaropus  annulatus,  life  history  of, 
789 

transmission  of  piroplasmidse  by,  789 
Marmorek's  serum  for  tuberculosis,  498 
Mastigophora,  definition  of,  721,  738 

polymastigina,   738 

protomonadina,  740 
Measles,  675 

investigation  for  virus  of,  675 
by  Hektoen,  675 
by  Home,  675 
Meat,   determination   of  nature  of,  by 
precipitin  tests,  254 

used  for  culture  media,  115 
soluble,  116 
Meat  extract,  116 
Meat-extract  agar,   127 
Meat-extract  broth,   124 
Meat-extract  gelatin,   126 
Meat-infusion  agar,  128 
Meat-infusion  broth,  124 
Meat-infusion  gelatin,  127 
Meat-poisoning  bacilli,  429,  475 
Meningitis,     microorganisms     causing, 
371 

primary,  371 

secondary,  371 

serum  therapy  of,  378 
Meningococcus.    See  Micrococcus  intra- 

cellularis  meningitidis 
Menozoitis,  760 
Metachromatic  granules,  11 
Metacresol  as  disinfectant,  77 
Metchnikoff's  therapy  of  gastrointesti- 
nal autointoxication  by  means  of 
Bacillus  bulgaricus,  716-717 

by  means  of  lactic-acid  bacilli,  717 
Mice,  method  of  injecting,  intravenous- 
ly, 173 
"Microbe  de  la  coqueluche,"  543 


Micrococci,    321.      See    also    Staphylo- 
cocci 
Micrococcus,  36 
Micrococcus  catarrhalis,  386 

differentiation    of,   from    gonococcus, 
386 
from  meningococcus,  386 
Micrococcus    intracellularis    meningiti- 
dis, 371 
cultivation  of,  374 
oxygen  in,  375 
viability  of  organism  in,  375 
differentiation  of,   from  Micrococcus 

catarrhalis,  386 
early  observation  of,  371-372 
immunization  against,  378 

agglutinins  in  immune  sera  in,  378 
modes  of  inoculation  of,  377 
morphology  of,  373 
parameningococcus  and,  379 
pathogenicity  of,  376 
in  animals,  377 
in  man,  376 
pseudomeningococcus       diflferentiated 

from,  379 
resistance  of,  376 
staining  of,  374 
susceptibility  of  animals  to,  377 
viability   of,   375 
Micrococcus  melitensis,  549 
cultivation  of,  550 
morphology  and  staining  of,  550 
Micrococcus  pharyngis  siccus,  387 
Micrococcus  tetragenus,  333 
cultivation  of,  333 
pathogenicity  of,  333 
Microorganisms,  discovery  of,  1 

pathogenic,  321 
Microphages,  definition  of,  276 
Microscopic  study  of  bacteria,  93 
in  fixed  preparations,  94 

process    of,    94.      See    also    under 
Staining 
in  living  state,  93 

by  hanging  block  method,  94 
by   hanging  drop   method,  93 
by  intravital  method  of  Nakanishi, 
94 


INDEX  OF  SUBJECTS 


835 


Microspira,  37 
Microsporon  furfur,  639 

clinical  picture  of  infection  of,  639 
cultivation  of,  640 
morphology  of,  639 
Milk,  alcoholic  fermentation  in,  702 
anthrax  bacilli  in,  707 
bacteria  in,  699 

butter    a    means    of    transmitting, 
711 

butter-making  aided  by,  711 

cheese-making  aided  by,  714 

numbers  of,  700 
estimating  of,  709 

propagation  of,  700 

sources  of,  699 

under    ordinarily    hygienic    condi- 
tions, 699 

varieties  of,  701 
"bitter,"  703 

butyric-acid  fermentation  in,  702 
certified,  701 
cholera  traced   to,   705 
coagulation   of  casein   in,  702 
color  changes  in,  703 
diarrheal  diseases  traced  to,  705 
diphtheria  traced  to,  705 
foot-and-mouth  disease  virus  in,  706 
lactic-acid  fermentation  in,  701 
pasteurization   of,   709 
pus  cells  and  leucocytes  in,  706 
relation    of,    to    infectious    diseases, 

703 
scarlet  fever  traced  to,  704 
"slimy,"  703 
streptococci  in,  705 
streptococcus    throat    infections    con- 
veyed by,  343,  706 
supervision  of  supply  of,  700 
tubercle  bacilli  in,  707 

infection  from,  708 

precautions  against,  708 
dairy  inspection  in,  708 

tuberculin  test  of  cows  in,  708 

transmission  of,  from  cow,  707 
typhoid    fever    epidemics    traced    to, 

703 
Milk  media,  130 


Milzbrand,  563 

Moeller's    method    of    staining    spores, 

98 
Moisture,   relation    of,   to   bacteria,   34 
Moitessier's  gas-pressure  regulator,  169 
Molds.    See  Hyphomycetes 
Monilia,  641 
Morax-Axenfeld  bacillus,  545 

cultivation  of,  546 

morphology  and  staining  of,  546 

pathogenicity  of,  547 
Motility  of  bacteria,  14 

by   fiagella,   14 

Brownian,   14 

effect  of  temperature  on,   15 

molecular,  14 

organs  of,  13 

true,  14 
Mucorinae,  reproduction  in,  635-636 
]\Iuguet.     See  Thrush 
Multiplicity  of  amboceptors  in  normal 
sera,  242 

of  complement  in  normal  sera,  242 
"Murrina  de  caderas,"  747 
Mycetoma,  clinical  picture  of,  627 

granules  in,  627 
melanoid,  627 

cultivation  of,  627-628 
morphology  of,   627 
ochroid,  627 
Mycomycetes,  635 

Nagana,  occurrence  of,  745 

parasite  of,  745 

transmission  of,  746 
Nakanishi,  "intravital"  staining  meth- 
od of,  in  study  of  bacteria,  94 
Negri    bodies   in    central    nervous   sys- 
tem in  rabies,  648 

demonstration  of,  648 

diagnosis  of  rabies  by,  650 

explanation  of,  650 

significance  of,  651 

staining  of,  108 
Neisser,   discovery  of  Diplococcus  gon- 
orrhoeae by,  380 

discovery  of  lepra  bacillus  by,  505 
Neosporidia.     See  Sarcosporidia 


836 


INDEX  OF  SUBJECTS 


Nephrotoxin,  201 

Neufeld    and    Rimpau's    discovery    of 

opsonic  substances,  282 
Neurotoxin,  201 
Neutral-red  medium  for   colon-typhoid 

differentiation,   138 
"New  tuberculin"    (Koch),  491 
"New     tuberculin-bacillary    emulsion," 

492 
Nitrate-solution  broth,  126 
Nitrifying  bacteria,  57 

action  of,  58 

agricultural  importance  of,  58 
Nitrogen,  for  preparation  of  anaerobic 

conditions,   151 
Nitrogen  fixation  by  bacteria,  54 
microorganism  of,  54 

in  root  tubercles,  55 
experimentation  on,  56 
microorganism  of,   55 
process  of,  56 

in  soil,  54 
Nitrogen  in  nutrition  of  bacteria,  28 

sources  of  supply  of,  28 
Noguchi's      modification      of     Wasser- 

mann  test  for  syphilis,  269 
Novy  jar,  154 

Nucleus  in  bacterial  cell,  10 
Nutrient  media.     See  Culture  media 
Nutrition  of  bacteria,  25 

carbon  in,  25 

hydrogen  in,  28 

nitrogen  in,  25 

oxygen  in,  25 

salts  in,  29 

Obermeier,    discovery    of    spirochsetes 

of  relapsing  fever  by,  605 
Obligatory  aerobes,  25 
Obligatory  anaerobes,  26 
Oi'dium  albicans,  640 

discovery  of,  640 

morphology  of,  640 

varieties  of,  640 
"Old  tuberculin"    (Koch),  491 
Opium  production,  bacteria  in,  719 
"Opsonic  coefficient  of  extinction,"  286 
Opsonic  index,  finding  of,  286 


Opsonic  test,  Wright's,  284. 

obtaining  of  bacterial  emulsion  for, 
284 
of  blood  serum  for,  284 
of  leucocytes  for,  284 
opsonic   index   in,    finding  of,   286 
parallel    control    test    on    normal 
serum  in,  285 
"pool"  in,  285 
technique  of,  284 
Simon,  Lamar,  and  Bispham's  tech- 
nique of,  286 
dilutions  in,  286 

opsonic  coefficient  of  extinction  in, 
286 
Opsonins,  281,  316 

decrease   of   phagocytic   power   upon 
introduction   of   bacteria   without, 
282 
definition  of,  282 
increase    of    phagocytic   power   upon 

introduction  of,  283 
Neufeld  and  Rimpau's  discovery  of, 

282 
normal  and  immune,  282-283 
specific  for  typhoid  bacillus,  424 
specificity  of,  282 
structure   of,    according   to   Hektoen 

and  Ruediger,  283 
Wright's  test  of,    See  Opsonic  test 
Wright's  theory  of,  282 
Orthocresol   as   disinfectant,   78 
Oscillaria  malariae,  764 
Osmotic  properties  of  bacterial  cell,  23 
Oxydases,  50 
Oxygen  as  disinfectant,  88 

in  development  of  bacteria,  25 
free,   26 

absence  of,  26 
indirect  supply  of,  26 
in  nutrition  of  bacteria,  25 
Oysters,  bacteriological  examination  of, 

718 
Ozone  as  disinfectant,  88 

Paltauf's     modification     of     Gram's 

stain,  103 
Pancreascytotoxin,  201 


INDEX  OF  SUBJECTS 


837 


Paper  used  in  filtering  culture  media, 

121 
Pappenheim-Saathof  stain,  of  diplococ- 

cus  gonorrhoeae,  381 
Paracolon  bacillus,  430 
Paracresol  as  a  disinfectant,  78 
Parameningococcus,  379 
Parasites,  bacterial,  29 
classification  of,  30 
facultative,  29 
media  for  growth  of,  29 
definition  of,  182 
infectiousness  of,  182 
pathogenicity  of,  182 
Paratyphoid  bacilli,  differentiation  of, 
on  sugar,  433 
types  A  and  B,  431 
Paratyphoid    fever,    differentiation    of, 

from  typhoid,  432 
Passive  immunity.     See  under  Immu- 
nity 
Passive     immunization,    definition    of, 

196 
Pasteur,  discovery  of  bacillus  of  chick- 
en cholera  by,  552 
discovery    of    bacillus    of    malignant 

edema  by,  468 
discovery  of  Diplococcus   pneumoniae 

by,  353 
technique  of,  in  rabies  therapy,  652 
Pasteurization  of  milk,  709 
Pathogenic  bacteria,  182,  321 
Pathogenic  protozoa.     See  Protozoa 
Pathogenicity,  fundamental  factors  of, 
181 
of  bacteria,   182 
Penicillium,  reproduction  in,  637 
Pepton-salt  solution  broth,  126 
Perez  bacillus  of  ozaena,  452 
Pericardial     exudates,     bacteriological 

examination  of,  175 
Peritoneal  exudates,  bacteriological  ex- 
amination of,  175 
Peritonitis    following    perforation     at- 
tributed to  colon  bacillus,  394 
Perlsucht,  498 

Pernicious   anemia   and   Bacillus    aero- 
geneS  capsulatus,  177 


Petri  dish,  115,  144 

Petruschky,  discovery  oi  Bacillus  fecsi!- 

lis  alkaligenes  by,  26,  427 
Pfeiffer,  discovery  of  influenza  bacillus 
by,  536 
discovery  of  Micrococcus  catarrhalis 

by,  386 
discovery  of  pseudo-influenza  bacillus 
by,  541 
Phagocytic  index,  286 
Phagocytosis,  275 
cells  active  in,  276 
"fixed,"  276 
macrophages,  276 
microphages,  276 
"wandering,"  276 
cells  of  animal  origin  in,  278 
chemotaxis  in,  277 
complement  or,  "cytase"  in,  279 
definition    of,   275 
dependence  of,  on  opsonins,  281-282 
diminution  of,  upon  introduction  of 
bacteria    without    opsonic    serum, 
282 
immune  body  or  "fixator"  in,  279 
immunity  and,  279 
in  higher  animals,  276 
in  protozoa,  275 
increase  of,  upon  the  introduction  of 

opsonic  substances  in  scrum,  282 
macrophages   in,  276 
Metchnikoff's  theory  of,  276 

opposition  to,  279 
microphages  in,  276 
process   of,  in  the  body  upon  intro- 
duction of  bacteria,  277 
upon    introduction    of    nutrient 
broth,  276 
susceptibility  of  various  microorgan- 
isms to,  278 
variety  of  phagocyte  in,  determined 
by  the  bacterium,  278 
Phenol  production  by  bacteria,  167 
Phosphates  in  the  nutrition  of  bacteria, 

29 
Phosphorescence  produced  by  bacteria, 

59 
Phragmidiothrix,  37 


838 


INDEX  OF  SUBJECTS 


Pliycomycetes,  635 

Pigment,  formation  of,  by  bacteria,  69 

chemical  nature  of,  59 

cultural  conditions  on,  60 
Piroplasmidae,    as    cause    of    Southern 

cattle  fever,    787 
clinical  observations  on,  788 
family  of,   787 
morphology  of,   787 
transmission  of,  788 
Pityriasis  versicolor,  639 
clinical  picture  of,  639 
microorganism  causing.     See  Micro- 

sporon  furfur 
occurrence  of,  639    . 
Plague,  bacillus  of,  554 

biology  of,  557 

control    of,    by    fumigation    with 
hydrocyanic  gas,  92 

degeneration  forms  of,  20 

immunization  against,  561 
active,  561 

involution  forms  of,  on  salt  agar, 
557 

isolation  and  cultivation  of,  556 

lesions  in  animals  produced  by,  559 

morphology  of,  555 

pathogenicity  of,  558 

relation  of  rats  to,  560 

resistance  of,  557 

staining  of,  556 

toxins  of,  561 

transmission  of,  560 

variations  in  virulence  of,  559 

viability  of,  557 
epidemics  of,  554 
in  animals,  558 

in  California  ground  squirrels,  560 

in  Manchurian  marmot,  560 

inoculation  in,  559 

spontaneous  infection  in,  559 
in  man,  558 

autopsy  findings  in,  558 

bacteriological  diagnosis  of,  in  life, 
558 

infection   in,  558 

localized  form  of,  558 

pneumonic  form  of,  558 


Plague,  transmission  of,  560 

Plague-like  disease  in  rodents,  562 

Planococcus,   36 

Planosarcina,  37 

Plasmodia,  differentiated  from  coccidia, 
766 
examination  of,  in  fresh  blood,  774 
finer  structure  of,  772 
form  of,  767 

Plasmodium  falciparum,  771 
Plasmodium  malarise,  770 
Plasmodium  vivax,  767 
life  cycle  of,  766 

Plasmodium     falciparum,     morphology 
of,   771 

Plasmodium  malarise,  765 

morphology   of,    770.     See   also  Ma- 
larial Parasites 

Plasmodium  praecox,  763 

Plasmodium  tenue,  787 

Plasmodium  vivax,  767,  786,  787 
morphology  of,  767 

Plasmolysis  of  bacterial  cell,  23 

Plasmoptysis  of  bacterial  cell,   24 

Plating  in  isolation  of  bacteria,  143 

Pleural  exudates,  bacteriological  exam- 
ination of,  175 

Pleuro-pneumonia,  organism  of,  542 

Plotz  bacillus,  cultivation  of,  678 

.    obtaining  of,  678 

Pneumobacillus.     See  Bacillus  mucosus 
capsulatus 

Pneumococcus,  discovery  of,  7 
different  types  of,  336 

Pneumococcus.     See  Diplococcus   pneu- 
moniae 

Pneumococcus-streptococcus  group,  mu- 
tation, 370 

Pneumonia,  complications   of,   362 
lobar,  etiology  of,  361 
infectiousness  of,  352,  361 

Poisons,  bacterial.     See  Bacterial  poi- 
sons 

Polar  bodies,  11 

special  stains  for,  107 

Poliomyelitis,  acute  anterior,  664 
immunity  in,  666 
infectiousness  of,    664 


INDEX  OF  SUBJECTS 


839 


Poliomyelitis,    inoculation    of    animals 

with  spinal  substance  of,  664 

by  Flexner  and  Lewis,  665,  666, 

667 
by  Knoepfelmacher,  665 
by     Landsteiner     and     Levaditi, 

665,  667 
by  Landsteiner  and  Popper,  665 
resistance  of  virus  of,  666 
Polychrome  stains   in  staining  of  bac- 
teria, 107 
Polymastigina,  definition  of,  738 
lamblia  intestinalis,  739 
tetramitis  mesuili,  739 
trichomonas,  738 
Potato  media,  130 

glycerin,  130 
Pour  plate,  technique  of  making,  145 
Precipitin  tests,  253 

bacterial  filtrates  for,  254 

technique  of,  255 
determining  nature  of  meat  by,  254 
precipitating  antisera  for,  253 
against  albumin  solutions,  253 
technique  of  production  of,  253 
protein  solutions  to  be  tested  by,  254 
Precipitins,  200,  235 

agglutinins  and,  structure  of    (Ehr- 
lich),  238 
cell-receptors  in,  238 
theoretical  considerations  concern- 
ing, 238 
bacterial  differentiation  by,  237 
differentiation    of    proteids    by,    237, 

254 
distinguishing  blood   of  animal    spe- 
cies by,  237 
effect  of  heat  on,   236 
experimentation  in,  235 
group  reaction  of,  237 
identity    of,   with    sensitizers,    236 
in  pneumococcus  immune  serum,  364 
in    streptococcus    immune    horse    se- 
rum, 347 
nature  of,  236 
specificity   of,   237 
Pressure,  relation  of,  to  bacteria,  34 
Proagglutinoids,  235 


Proteid   differentiation  by   complement 
fixation,  273 
substances  necessary   for,  273 
technique  of,  274 
by  precipitins,  237,  254 
Proteid  injections,  anaphylaxis  in,  295 

See  also  Anaphylaxis 
Proteids  in  bacterial  cell,  22 
Protein  solutions   for   precipitin  tests, 

preparation  of,  254 
Proteins,  bacterial,    186 
Proteolytic  enzymes,  43 
action  of,  44 

in  breaking  down  animal  excreta, 
46 
bacteria  producing,  44 
proteids  necessary  to,  44 
ptomains  produced  by,  45 
Proteosoma  (plasmodium)  praecox,  763 
Proteus  group,  bacilli  of,  454,  455 
cultivation  of,  454 
morphology  and  staining  of,  454 
occurrence  of,  454 
pathogenicity  of,  455 
Protomonadina,  740 
bodonidffi,   740 
prowazekia,  740 
cercomodidse,   740 
Cercomonas  hominis,   740 
definition  of,  740 
trypanisomidae,  740 
Protozoa,   classification  of,   721 
definition  and  functions  of,  721 
infusoria   (ciliata),  792 
mastigophora    (flagellata),   721,   738 
sarcodina    (rhizopoda),  721,  723 
sporozoa,  722,  760 
technique    of    blood    examinations 
for,  795 
Protozoa    and   bacteria,   differentiation 
of,  1 
staining  of,  108 
Prowazekia,  740 

Pseudo-dysentery  bacillus,  437,  438 
Pseudo-influenza  bacillus,  541 
Pseudo-membranes  in  diphtheria,  518 
Pseudoraeningococcus,  379 
Pseudomonas,  37 


840 


INDEX  OF  SUBJECTS 


Ptomains,  45,  185,  306 

bacterial  poisons  and,   185 

discovery  of,  185 

occurrence  of,  185 

toxins  distinguished  from,  45 

varieties  of,  45 
Pus,     bacteriological     examination     of, 

175 
Pus  cells  and  leucocytes  in  milk,  706 
Putrefaction,  action  of,  44 
Putrefactive  bacteria,  quantitative  an- 
alysis of,  21 
Putrescin,  45 

Pyemia,  definition  of,   184 
Pyocyanase,  580 

immunizing  powers  of,  580 
Pyocyanin,  578 
Pyocyanolysin,  581 
Pyrogallic   acid,  use  of,  in  cultivation 

of  anaerobic  bacteria,  152 


Quartan  fever,  parasite  of,  770 

symptoms  of,   776 
Quinine,  blackwater  fever  precipitated 

by,  786 


Rabies,  646 
course  of,  647 
in  animals,  647 
in  men,  648 
cultivation  of  organism  of,  by  Nogu- 

chi,  651 
diagnosis    of,   by    presence   of   Negri 
bodies   in  central  nervous   system, 
648-650 
experimental   infection   of,   646 
incubation  in,  647 

Negri  bodies  in  central  nervous  sys- 
tem, 648 
demonstration  of,  648 

by  Van  Gieson's  method,  649 
by  Williams  and  Lowden's  meth- 
od, 650 
staining  in,  649 

Mann's  method  of,  649 
diagnosis  by,   649-650 


Rabies,  occurrence  of,  646 
pathology   of,    648 

specific  therapy  of    (Pasteur's   tech- 
nique), 652 
attenuation     and     preparation     of 

virus  fixe  in,  652 
inoculation   of   rabbits   with   virus 

fixe  in,  652 
spinal    cord    of   inoculated    rabbits 
in  desiccation  of,  653 
emulsification   of,   654 
treatment  of  cases  with  injections 
of  spinal-cord  solution  in,  654 
Hogyes  dilution  method  in,  656 
scheme  of,  used  at  Pasteur  In- 
stitute, 654 
used  in  New  York  Department 
of  Health,  655 
virulence  of  virus  of,  647 
Racial  immunity,  191 
"Rage."     See  Rabies 
Rat,  relation  of,  to  plague,  560 
Rat-bite  fever,  617 
Rat  leprosy,  510 

relation  of,  to  human  leprosy,  511 
Rauschbrand.     See  Bacillus  of  sympto- 
matic anthrax 
Receptors    of   toxin    molecule    in    side- 
chain  theory,  213 
chemical  action  of,  213 
over-production  of,  214 
Red  blood  cells,  antibodies  produced  by, 

200 
Reducing  powers  of  bacteria,  167 
Refractive  index   of  parts  of  bacterial 

cell,  24 
Reichel  filter,  122 

Reichert's  thermo-regulator,  158,  159 
Relapsing  fever,  605 
immunity  in,  610 
symptoms  of,  608 
transmission  of,  610 
varieties  of,  609 
Relapsing  fever  spirochsete,   605 
cultivation  of,   606 
morphology  and  staining  of,  605 
pathogenicity  of,  608 
in  animals,  608 


INDEX  OF  SUBJECTS 


841 


Relapsing  fever  spirochaete,  pathogenic- 
ity of,   in  man,  608 
symptoms  of,  608 
transmission  of,  610 
varieties  of,  609 
Reproduction   of  bacteria,   17 
Resistance  of,  definition  of,  189 
Rhinoscleroma,  bacillus  of,  451 
Rhizopoda.    See  Sarcodina 
Ricin,  experimentation  with,  204 
Ringworm,  643 
Rodents,  destruction  of,  by  hydrocyanic 

gas,  92 
Root  tubercles,  55 

microorganism    of   nitrogen    fixation 
in,  55 
Roux's  method  of  anaerobic  cultivation 

of  bacteria,  149 
Russell's  double  sugar  agar  for  colon- 
typhoid  differentiation,  138 

Saccharomycetes.      See    Yeasts    and 

Yeast  cells 
Salts  in  nutrition  of  bacteria,  28 
Saprophytes,  bacterial,  29,  30 

definition  of,  182 
Sarcina,  36 

Sarcodina,  amebae,  723 
Sarcophysematous  bovis.     See  Bacillus 

of  symptomatic  anthrax 
Sarcosporidia,  791 
Scarlatina.    See  Scarlet  fever 
Scarlet  fever,  676 

favorable    influence   of    streptococcus 
antisera  in,  676 

streptococci  present  in,  676 

traced  to  milk,  704 
Schaudinn  and  Hoffmann,  discovery  of 

Spirochaeta  pallida  by,  594 
Schizomycetes,  36 
Schizonta,  760 
Schizotrypanum  cruzi,  755 
Schweineseuche,  553 
Scorpion  poison,  antitoxin  for,  199 
"Sensitization"  of  antigen,  241 
Septicemia,  definition  of,  184 

diagnosis  of,  by  isolation  of  bacteria 
from  the  blood,  178 


Septicemia,  due  to  colon-bacillus  infec- 
tion, 394 
hemorrhagic.     See  Hemorrhagic  sep- 
ticemia 
Serum  media,  131 

Loeffler's,  131 
Serum  reactions,  technique  of,  250 

See  also  under  individual  tests 
agglutination  tests  in,  251 
antigen  determined  in,  by  comple- 
ment fixation,  271 
for  typhoid  fever,  271 

obtaining  of  material  for,  271 
test  in,  272 
bactericidal  and  bacteriolytic  tests 

in,  255 
complement  fixation  in,  for  deter- 
mination of  antibodies,  262 
for    determination    of    antigen, 

271 
for  proteid  differentiation,  273 
hemolytic  tests  in,  259 
precipitin  tests  in,  253 
proteid  differentiation  by  comple- 
ment fixation  in,  273 
substances  necessary  for,   273 
test  in,  274 
Wassermann  test  in,  263 
modifications  of,  268 
"Serum  sickness,"  296 
Serum   water   media   for    fermentation, 

tests,  132 
Shick   reaction,   for   determining  pres- 
ence of  diphtheria  antitoxin  in  blood, 
526 
Shiga,  discovery  of  dysentery  bacillus 

by,  435 
Shiga's  bacillus,  435 

cultural  characteristics  of,  436 
morphology  of,  435 
Side-chain    theory    of    toxin-antitoxin 
reaction,  212 
chemical  action  in,  213 
elements  of  molecules  in,  213 
atom  group,  213 
side  chains  or  receptors,  213 
over-production  of  receptors  in,  214 
Side  chains,  action  of,  in-Ehrlich's  the- 


842 


INDEX  OF  SUBJECTS 


ory  of  lytic  process  in  blood  serum, 
226 
Slanting  of  culture  media,  123 
Sleeping    sickness.      See    Trypanosomi- 
asis 
"Slimy"  milk,  bacteria  causing,  703 
Smallpox,   657 

etiological  factor  of,  657 
immunization  in,  658 
by  vaccination,  659,  663 
Jenner's  discovery  of,  659 
technique  of,  663 
value  of,  663 
production  of  vaccine  for,  647.   See 
also  under  Vaccine 
occurrence  of,  657 
protozoan   incitant   of,    research   for, 

657 
relation  of  chicken-pox  to,  660 
relation  of  cowpox  to,  659 
transmission  of,  658 
vaccine  bodies   in,   discovery  of,  657 
explanations  for,  658 
Ewing's,  658 
Smegma  bacillus,   502,   503,  594 
cultivation  of,  504 
morphology  of,  503 
occurrence  of,  503 
staining  of,  504 

identification  of  bacillus  by,  504 
tubercle  bacillus  and,  differentiation 
between,  by  stains,  106 
Smith's  modification  of  Pitfield's  meth- 
od of  staining  flagella,  101 
Snake  poison,  antitoxin  for,  199 
Soil,  bacteria  in,  685 

from  burial   of  infected  cadavers, 

687 
in  agricultural  regions,  685 
numerical  estimation  of,  687 
pathogenic,   in  surface  layers,  686 
Solutions,    saturated,    for    staining    of 
bacteria,  95 
staining-power  of,  96 
Soor.     See  Thrush 

Southern  cattle  fever,  parasite  of,  787 
Species  immunity,  190 
differences  in,  190 


Specific  gravity   of  forms  of  bacterial 

cell,  24 
"Specific  precipitates,"  235-236 
Spider  poison,  antitoxin  for,  199 
Spinal    fluid,    bacteriological    examina- 
tion of,   176 
Spirillace8e„37 
Spirillum,  37 

description   of,   9 
Spirillum  cholerae  asiaticse.     See  under 

Cholera 
Spirillum  Deneke,  591 
Spirillum  of  Finkler-Prior,  539 
Spirillum  Massaua,  591 
Spirillum  Metchnikovi,  590 
Spirochseta,  genus,  37 
Spirochseta  anserina,  616 
Spirochaeta  calligyrum,  617 
Spirochseta  Duttoni,  610 
Spirochaeta  gallinarum,  615 

cultivation  of,  by  Noguchi,  616 

immunization  against,  616 

similarity    of,    to   Spirochaeta   anser- 
ina, 616 

transmission  of,  615,  616 
Spirochaeta  icterohsemoragica,   617 
Spirochaeta  macrodentium,   617 
Spirochaeta  microdentium,  617 
Spirochaeta  pertenuis,  614 

morphology  of,  615 

similarity  of,  to  Spirochaeta  pallida, 
615 
Spirochaeta  phagedenis,  616 
"Spirochaeta  refringens,"  595 
Spirochaete  of  relapsing  fever.    See  Re- 
lapsing fever  spirochaete 
Spirochaete   of   Vincent's    angina.      See 

Vincent's  angina,  spirochaete  of 
Spirochaetes,  cultivation  of,  593 

differentiation  of,  from  spirilla,  593 

diseases    caused    by,    592.     See   also 
under  specific  names 

reproduction  in,  592 

structure  of,  592 
Spirosoma,  37 
Splenomegaly,  infantile,  758 
Spore    stains    in    staining   of   bacteria, 

97 


4 


INDEX  OF  SUBJECTS 


843 


Spores,  bacterial,   15 
formation  of,  15 
germination  of,  17 
position  of,    17 
relation  of  temperature  to  cultures 

with,  33 
varieties  of,  16 
arthrospores,  16 
true  or  endospores,  16" 
vegetative  forms  from,  17 
Sporonta,  760 
Sporotrichosis,  644 
Sporozoa,   hemosporidia,   760 
sarcosporidia,  791 
subclasses  of,  neosporidia,  791 
telosporidia,  760 
Sporulation,    physiological   significance 
of,  17 
process  of,   16 
Sprue,  tropical,  640 
Sputum,  disinfection  of,  87 
Stable  antitoxin,  206 
Staining  of  bacteria,   chemical   princi- 
ples in  process  of,  96 
acid-fast  bacteria  stains,  104 
Baumgarten's  method,  106 
Bunge   and  Trautenroth's  meth- 
od, 106 
Ehrlich's  method,  104 
Gabbet's  method,  105 
Pappenheim's  method,   106 
Ziehl-Neelson  method,  105 
capsule  stains,  98 
Buerger's  method,  99 
Hiss'  methods,  98 

copper  sulphate,  98 
Huntson's  method,  98 
Wadsworth's  method,  99 
Welch's  method,  98 
differential  stains,  102 
Gram's  method,  102 
classification  by,  104 
Paltauf's  modification  of,  103 
Sterling's  modification   of,  103 
flagella  stains,  100 
Loeffler's  method,  100 
Smith's  modification  of  Bitfield's 
method,  101 


Staining  of   bacteria,   chemical   princi- 
ples in  process  of,  flagella  stains, 
Van  Ermengem's  method,  101 
polychrome  stains,  107 
Giemsa's  method,  108 
Jenner's  method,  108 
Wood's  method,   109 
Wright's  modification  of  Leish- 
man's  method,  108 
special  stains  for  polar  bodies,  107 
Neisser's  method,  107 
Roux's  method,  107 
spore  stains,  97 

Abbott's  method,  97 
Moeller's  method,  98 
staining  in  tissues,  110 

for  actinomyces  in  sections,  112 
for  Gram-positive  bacteria,  111 
Gram-Weigert  method,  111 
in  celloidin  sections,  111 
in  paraffin  sections.  111 
for   tubercle  bacilli   in   sections, 
112 
in  celloidin  sections,  112 
in  paraffin  sections,  112 
Loeffler's  method,  112 
saturated  solutions  used  in,  95 
staining  solutions  in,  power  of,  96 
steps  in  process  of: 

( 1 )  smearing,  94 

(2)  drying,  95 

(3)  fixing,  95 

(4)  staining,  95 

(5)  washing,  95 

(6)  blotting,  95 

(7)  mounting,  95 
Standardization     of     diphtheria     anti- 
toxin, 218 

of  tetanus  antitoxin,  221 
Staphylococci,  321.    See  also  under  in- 
dividual staphylococci 

definition  of,  321 

in  feces,  177 
Staphylococcus  epidermidis  albus,  332 
Staphylococcus  pyogenes  albus,  332 
Staphylococcus  pyogenes  aureus,  322 

cultural  characters  of,  323 

immunization  against,  331 


844 


INDEX  OF  SUBJECTS 


Staphylococcus  pyogenes  aureus,  immu- 
nization against,  active,  332 
agglutinins  in,  331 
modes  of  inoculation  with,  327 
morphology  of,  322 
pathogenicity  of,  326 
in  animals,  327 
in  man,  327 
pigment  formation  of,  325 
resistance  of,  325 
to  chemicals,  326 
to  desiccation,  326 
to  heat  and  cold,  325 
staining  of,  322 

susceptibility  of  animals  to,  326 
susceptibility  of  man  to,  327 
thermal  death  point  of,  325 
toxic  products  of,  328 
endotoxins,  328 
hemolysins,   328 

leucocidin,    329.      See   also    under 
Leucocidin 
virulence  of,  326 
Staphylococcus  pyogenes  citreus,  332 
Steam  in  sterilization,  67 
live,  69 
saturated,  68 
superheated,  68 
Stegomyia  fasciata,  673 
Sterilization  of  culture  media,  121 
filtration  in,  122 
heat  in,  121 
Sterling's  modification  of  Gram's  stain, 

103 
Sternberg,     discovery     of    Diplococcus 

pneumoniae  by,  353 
Stimulins,  281 

Streaking,  surface,  in  isolation  of  bac- 
teria, 148 
"Street  virus,"  647 
Streptococci,  36,  335 

capsulated,  description  of  organisms 

reported  as,  367-369 
classification  of,  347 

by  Andrews  and  Horder,  349 
by  carbohydrate  fermentation  pow- 
ers, 348    ' 
by  reactions 'to  immune  sera,  349 


Streptococci,  classification  of,  morpho- 
logical, 348 
Streptococcus    longus     seu     ery- 

sipelatos  in,  348 
Streptococcus  minor  seu  viridans 

in,  348 
Streptococcus  mucoeus  in,  348 
definition  of,  335 

diflferentiation  of,  from  pneumococci, 
357,  367 
cultural,  368  m|{ 

morphological,  367  •'^ 

epidemic  throat  infections  by,  343 
in  feces,  177 

in  milk,  705  ! 

in  milk  epidemics,  343 
predilection   of,    for   definite   tissues, 

351 
preparation  of,  for  agglutination  test, 

251 
pyogenic.     See    Streptococcus    pyog- 
enes 
Streptococcus  anginosus,  349 
Streptococcus  equinus,  349 
Streptococcus  erysipelatis,  342 
Streptococcus  equinus,  349 
Streptococcus   longus   seu  erysipelatos, 

348 
Streptococcus     mitior     seu     viridans, 

348 
Streptococcus  mucosus,  350,  351 
Streptococcus  pyogenes,  335 
brevis,  337,  338 
cultivation  of,  337 
early  experimentation  with,  335 
immunization  against,  345 

immune    sera  of   infected   animals 
in,  545 
agglutinins  in,  347 
precipitins  in,  347 
specificity  of,  347 
standardization  of,  347 
leucocyte  extracts  in,  347 
technique  of,  346 
longus,  337 

modes  of  inoculation  with,  341 
in  animals,  341 
in  man,  342 


INDEX  OF  SUBJECTS 


845 


Streptococcus     pyogenes,      morphology 
of,  337 

pathogenicity  of,  340 
in  animals,   340 
in  man,  342 

resistance  of,  339 

staining  of,  337 

susceptibility  of  animals  to,  341 

toxic  products  of,  344 
endotoxins,    344 
hemolysins,  344 

virulence  of,  340 
Streptococcus  salivarius,  349 
Streptothrix,  37,  619 

cultivation  of,  621 

morphology  of,  619,  621 
Sublethal  doses  of  virulent  bacteria  in 

active  immunization,  195 
Suctoria.     See  Infusoria 
Sugar-free  broth,  125 
Sulphates  in  nutrition  of  bacteria,  29 
Sulphur  bacteria,  60 

physiology  of,  61 

spectroscopic  examination  of,  61 

varieties  of,  60 
Sulphur    dioxid     as    disinfectant,    88, 

89 
Sulphuretted  hydrogen.     See  Hydrogen 

sulphid 
Suprarenal  cytotoxin,  201 
Surra,  clinical  course  of,  745 

occurrence  of,  745 

parasite  of,  745 

transmission  of,  745 
Swine-plague  bacillus,  553 

diflferentiation    of,    from    hog-cholera 
bacillus,  554 

immunization  against,  553 

morphology  of,   553 

pathogenicity  of,  553 
Symbiosis  of  bacteria,  30,  31 
Symptomatic  anthrax,  bacillus  of.     See 

Anthrax,  symptomatic 
Syphilis,  593 

in  monkeys,  601 

in  rabbits,  602 

microorganism    of.      See   Spircchseta 
pallida 


Tanning  of  hides,  bacteria  in,  720 
Telosporidia.     See  Hemosporidia 
Temperature,    attained   by    application 
of  various  degrees  of  pressure,  72 
effect  of,  on  activity  of  bacteria,  15 
high,  34 
low,  34 
relation  of,  to  bacteria,  31 
maximum,  31 
minimum,  31 
optimum,  32 
to  cultures  with  spores,  33 
to  vegetative  forms,  33 
Tertian  fever,  parasite  of,  767 

symptoms  of,  776 
"Tetanolysin,"  205,  464 
"Tetanospasmin,"  463 
Tetanus  antitoxin,  220 
production  of,  220 
horses  used  in,  221 
technique  of,  221 
toxin  for,  220 
standardization  of,  222 
therapeutic  value  of,  464 
unit  of  ( Society  of  American  Bacteri- 
ologists), 222 
Tetanus  bacillus,  456 

autopsy  findings  in  infections  of,  460 

biological  characteristics  of,  458 

cultivation  of,  458 

distribution  of,  457 

early  observation  of,  456 

favorable   conditions   for  growth   of, 

459 
incubation  of,  460 
isolation  of,  by  Kitasato,  456 
morphology  of,  456 
pathogenicity  of,  459 
following  wounds,  460 
relation  of  spores  to,  459 
resistance  of,  459 
staining  of,  457 
toxin  of,  460 

central    nervous    system    attacked 
by,  463 
mode  of  reaching,  463 
incubation  period  of,  463 
isolation  of,  461 


846 


INDEX  OF  SUBJECTS 


Tetanus  bacillus,  toxin  of,  isolation  of, 
by  chemical  reaction,  462 
by  filtration,  461 
by  precipitation,  461 
production  of,  461 
resistance  of,  462 
strength  of,  462 
susceptibility  of  animals  to,  462 
Tetanus    spores,   transportation   of,    to 

organs,  460 
Tetramitis  mesnili,  739 
Thermal  death  points,  33 
Thermo-regulators  for  incubators,  159 
Lautenschlager's,   158,   159 
Reichert,  158,  159 
Thiothrix,  38 
Thrush,  640 

microorganism  causing,  640 
Tick.    See  Margaropus  annulatus 
Timothy,  bacillus  of,  502 
Tissue  sections,  method  of  staining,  111 
Gram-Weigert,  111 
in  celloidin  sections.  111 
in  paraffin  sections.  111 
staining  of  bacteria  in,  110 
Titration  of  culture  media,  117 
color  indicator  in,  117 
process  of,  117 

for  alkaline  media,  118 
reaction  of,  117 
adjustment  of,  119 
Tobacco  industry,  bacteria  in,  719 
Toxin,  constitution  of  (Ehrlich),  210 
graphic  form  of  (Ehrlich),  211 
■  views  of  Arrhenius  and  Madsen  on, 

212 
diphtheria.     See     under     Diphtheria 

toxin 
endotoxin  distinguished  from,  186 
epitoxoid  in,  208 

in  side-chain  theory,  cell-nutrition  in, 
213 
chemical  action  of,  213 
elements  of,  213 
atom  group,  213 
side  chains  or  receptors,  213 
over-production  of  receptors  in,  214 
molecule  of,  haptophore  group  in,  207 


Toxin,  molecule  of,  toxophore  group  in, 
207 
partial  absorption  of,  209 
standardization  of,  207 
Limes  death  in,  208 
Limes  zero  in,  207 
time  changes  in,  206 
toxoid  form  of,  207 
protoxoids  in,  209 
syntoxoids  in,  209 
toxon  and,  difference  in  action  of,  209 
toxon  in,  209 
used    for    production    of    diphtheria 

antitoxin,  216 
valency  of  antitoxin  for,  210 
Toxin-antitoxin  reaction,  203 
side-chain  theory  in,  212 
summary  of,  215 
theories  as  to  process  of,  203 

by  destruction  of  toxin  by  its  spe- 
cific antitoxin,  203 
by  direct  union  of  toxin  and  anti- 
toxin, 203 
through  mediation  of  tissue  cells, 
203 
time  element  in,  204 
Toxin  solution,  normal,  205 
Toxin  unit,  205 
Toxins,  185 
•   compared  with  pigments,  186,  309 

summary  of,  305 
Toxoid  form  of  diphtheria  toxin,  207 
Toxoids,  varieties  of,  209 
epitoxoid  form  in,  208 
protoxoids,  209 
syntoxoids,  209 
Toxon,  in  diphtheria  toxin,  209 

toxin  and,  difference  in  action  of,  209 
Toxon  molecule,  209 

haptophore  group  in,  209 
toxophore  group  in,  209 
Toxophore  group  in  toxin  molecule,  207 

in  toxon  molecule,  209 
Trachoma,  hemoglobinophilic  bacilli  in. 

541 
Treponema  marsus  muris,  017 
Treponoma  pallidum,  593.  617 
animal  pathogenicity  of,  601 


INDEX  OF  SUBJECTS 


847 


Treponema    pallidum,    cultivation    of, 
599 
dark-field  examination  of,  597 
by  Muhlens,  600 
by  Noguchi,  600 
demonstration  of,  596 
in'  living  state,  596 
in  smears,  597 

by  India-ink  preparation,  598 
by     Schaudinn     and     Hoffman's 

method  of  staining,  597 
by   Wood's   method  of   staining, 
597 
in  tissues,  598 

by  Levaditi's  method,  598 
by    Levaditi    and    Manouelian's 
method,  599 
immunization  against,  603 
active,  603 
passive,  603 
infection  of  animals  by,  601,  602 
of  cornea  of  rabbits,  602 
of  tests,  of  rabbits,  602 
morphology  of,  595 
observation  of,  596-597 
occurrence  of,  in  syphilis  cases,  595- 

596 
staining  of,  108 

Fontana's  method  for,  598 
Trichomonas,  738 
Trichomonas  intestinalis,  738 
Trichomonas  vaginalis,  738 
Trichomycetes.     See  Chlamydobacteria- 

ceae 
Trichophyton  tonsurans,  643 
cultivation  of,  644 
demonstration  of,  643 
morphology  of,  643 
occurrence  of,  643 
Tricresol,  78 
Trillat  autoclave,  89 
Tropical  sprue,  640 
Trypanosoma  avium,  750 
Trypanosoma  brucei,   as   cause  of  na- 
gana,  745 
cultures  of,  747 
morphology  of,  746 
transmission  of,  746 


Trypanosoma  equiperdum,  as  cause  of 
dourine,  748 
occurrence  of,  748 
site  of,  749 
Trypanosoma  evansi,  as  cause  of  surra, 
745 
morphology  of,  745 
Trypanosoma    gambiensi,    animal    host 
of,  753 
as  cause  of  trypanosomiasis,  751 
morphology  of,  753 
pathogenicity  of,  753 
Trypanosoma  hippicum,  disease  due  to, 
747 
incubation  period  of,  747 
morphology  of,  748 
prophylaxis  against,  748 
Trypanosoma  lewisi,  immunity  to,  743 
insect  hosts  of,  744 
morphology  of,  744 
multiplication  of,  744 
occurrence  of,  743 
transmission  of,  743 
Trypanosoma  rhodesiense,  754 
Trypanosoma  rotatorium,   cultures   of, 
743 
morphology  of,  742 
multiplication  of,  743 
occurrence  of,  742 
Trypanosomiasis,  clinical  signs  of,  752 
diagnosis  of,  754 
etiology  of,  752 
mortality  from,  751 
prophylaxis  against,  754 
symptoms  of,  751 
treatment  of,  754 
Trypanosomidae,  cultivation  of,  742 
history  of,  740 
morphology  of,  741 
species  of,  740,  741 
transmission  of,  741 
Tubercle  bacillus,  479 
bacilli  related  to,  498 
Bacillus  butyricus,  502 
bacillus  of  avian  tuberculosis,  500 
cultivation  of,  500 
discovery  of,  500 
morphology  and  staining  of,  500 


848 


INDEX  OF  SUBJECTS 


Tubercle  bacillus,  bacilli  related  to,  ba- 
cillus of  avian  tuberculosis,  sus- 
ceptibility of  animals  to,  500 
bacillus  of  bovine  tuberculosis,  498 
cultivation  of,  499 
diflferentiation   of,   from   human 

type,  499 
early  investigation  of,  498 
morphology  of,  499 
bacillus  of  fish  tuberculosis,  495 
bacillus  of  leprosy,  502,  509 
bacillus  of  timothy,  502 
bacillus  of  turtle  tuberculosis,  501 
Bacillus  smegmatis,  502 
biological  considerations  of,  486 
chemical  analysis  of,  490 
cultivation  of,  483 
media  for,  484 
"Nahrstoff  Heyden"  in,  485 
discovery  of,  7 
early  investigation  of,  479 
examination  for,  by  animal  inocula- 
tions, 175 
by  Ziehl-Neelsbn  staining  method, 
176 
in  circulating  blood,  489 
in  feces,  178 
in  milk,  709 
isolation  of,  483 

Petroff's  method  of,  484 
leprosy   bacillus   and,   differentiation 

between,  by  stains,  106 
methods  of  staining,  104,  105,  106 
in  sections,  112 
celloidin,  112 
paraffin,  112 
morphology  of,  479 
Much  granules,  482 
pathogenicity  of,  486 
frequency  in,  487 
mode  of  infection  in,  487 
mortality  in,  487 
preparation     of,     for     agglutination 

test,  251 
quantitative  analysis  of,  22 
smegma  bacillus  and,  differentiation 

between,  by  stains,  106 
staining  of,  480 


Tubercle    bacillus,    staining    of,    differ- 
entiation     of,      from      acid-fast 
group   by  Pappenheim's   method 
of,  482 
Ehrlich's     anilin-water-gentian -vio- 
let solution  in,  481 
Gabbet's  decoloration  and  counter- 
staining  in,  481 
Ziehl's    carbol-fuchsin   solution  in, 
481 
toxins  of,  490 

endotoxins  in,  490 
tuberculins  in,  490 

bouillon  filtre   (Denys),  492 
"new    tuberculin-bacillary   emul- 
sion"  (Koch),  492 
"new  tuberculin"   (Koch),  491 
original  method  of  making  of, 

491 
present  method  of  making  of, 
491 
"old  tuberculin"  (Koch),  491 
"tuberculoplasmin"         ( Buchner 
and  Hahn ) ,  492 
use  of  antiformin  in  examination  for, 
483 
Tuberculin.     See  under  Tubercle  bacil- 
lus, toxins  of 
"Tuberculoplasmin"       ( Buchner       and 

Hahn),  492 
Tuberculosis,    complement    fixation    in, 
494 
frequency  of,  486 
human  and  bovine  types  of  bacilli  in, 

in  infections  of  man,  488 
immunization  in,  passive,  497 
Maragliano's  serum  in,  498 
Marmorek's  serum  in,  498 
mode  of  infection  in,  487 
mortality  of,  486 

tuberculin  in,  diagnostic  use  of,  493 
cutaneous  reaction  in,  494 
in  cattle,  495 

ophthalmo  reaction  in,  494 
subcutaneous  injection  of,  493 
dosage  and  reaction  in,  493 
therapeutic  uses  of,  496 
original,  496 

# 


INDEX  OF  SUBJECTS 


849 


Tuberculosis,  tuberculin  in,  therapeutic 
uses  of,  present,  497 
dosage  in,  497 

preparations  employed  in,  497 
Tubing  of  culture  media,  121 
Typhoid  bacillus.     See  under  Typhoid 

fever 
Typhoid  carrier  state  in  rabbits,  404 
Typhoid  carriers,  412,  415 
Typhoid  fever,  bacillus  of,  399 

antigenic  properties  of,  421,  426 

bacteriemia  in,  405 

biological   conditions   favorable  to, 

403 
cultivation  of,  399 
cultural     differences     within     the 

group,  403 
differentiation    of,    from     Bacillus 
fecalis  alkaligenes,  427 
from   meat-poisoning   and    para- 
typhoid bacilli,  428 
discovery  of,  7,  399 
immunization  against.     See  under 
Typhoid  fever,  immunization  in 
In  blood  during  disease,  405 
obtaining  cultures  of,  405 
in  feces,  177 
in  gall-bladder,  411 
in  rose  spots,  412 
in  sputum,  412 
in  stools,  406 

examination  in,  406 
isolation  of,  407 

on  Conradi-Drigalski  medium, 

408 
on  Eisner's  potato-extract  gel- 
atin, 407 
on  Endo's  fuchsin-agar,  409 
on    Hiss'    agar-gelatin    media, 

407 
on    Loeffler's    malachite-green 
media,  409 
time  of  appearance  in,  406 
in  urine,  411 
•in  water,  694 
inoculation  of  animals  with,  404 

with  endotoxin  of,  417 
morphology  of,  399 


Typhoid  fever,  bacillus  of,  pathogenic- 
ity of,   404 
in  animals,  404 
in  man,  404 
staining  of,  339 

suppurative  lesions  due  to,  412 
toxic  products  of,  415 
obtaining  of,  417 
varieties  of: 
endotoxins,  415 
true  toxins,  416 
typhoplasmin,  416 
diagnosis  in,  by  agglutinins  in  blood 
serum,  420 
Widal  test  in,  421 

obtaining  blood  for,  422 
by  bactericidal  substances  in  blood 

serum,  419 
by  bactericidal  tests  in  vivo,  258 
by  opsonic  index,  424 
epidemics  of,  traced  to  milk,  703 
hygienic  considerations  in,  413 
immunization  in,  417 

by  inoculation  with  typhoid  bacilli, 
417 
active,  424 

technique  of  Pfeiffer  and  Kolle 
in,   425 
of  Wright  in,  425 
substances  found  in  blood  after, 
418 
agglutinins  in,  419 
chief  or  major,  420 
group,  420 
bactericidal,  419 
bacteriolytic,  418 
opsonins  in,  424 
precipitins   in,  423 
obtaining  blood  cultures  in,  178 
prophylactic  measures  in,  414 
prophylactic  vaccination  in,  426 
specific  therapy  in,  424 
transmission  of,  413,  414 
by  flies,  415 
from  milk,  414 
from  oysters,  410 
from  water  supply,  414 
without  intestinal   lesions,  413 


850 


INDEX  OF  SUBJECTS 


"Typhoplastiliin,"  416 

Typhus  fever,  bacillus  of  Plotz,  678 

bacillus  of  Ricketts  and  Wilder,  677 

distribution  of,  677 

etiology  of,  677 

identity  of,  with  Brill's  disease,  677 

inoculation  of  animals  with,  677 

transmission  of,  679 

Urine,  bacteriological  examination  of, 

176 
Urobacillus  liquefaciens,  455 
Uschinsky's  proteid-free  medium,  126 

Vaccine  production,  for  immunization 
in  smallpox,  660 
animals  used  in,  660 
calves  used  for,  660 

cleanliness  observed  in  stabling  of, 

660 
material   used   for  vaccination  of, 
661 
'    vaccination  in,  661 

preparation  of  field  in,  661 
scarifications  in,  661 
vaccinia    vesicles    developed    in, 
661 
obtaining  of  vaccine  from,  662 
by  curettage,  662 
by  ivory  tips,  662 
testing  of  vaccine  in,  for  bacteria,  662 
for  efficiency,  662 
Vaccine  therapy  of  Wright,  286 
dosage  for,  288 
opsonic  curve  in,  288 
production  of  vaccines  in,  286 
standardization  of  emulsion  in,  287 
enumeration    of    bacteria    against 

red  blood  cells  in,  287-288 
sterilization  of  vaccine  in,  288 
Vahlkampfia,  723 
Van   Ermengem,   discovery  of  Bacillus 

botulinus  by,  476 
Van    Ermengem's    method    of   staining 

flagella,  101 
Variola.     See  Smallpox 
Vegetative  forms  from  bacterial  spores, 
17 


"Vibrion     septique."       See    Malignant 

edema,  bacillus  of 
Vincent's  angina,  610 
spirochaete  of,  611 
cultivation  of,  613 
fusiform  variety  of,  612 

bacilli  of  other   diseases  resem- 
bling, 613.    See  also  under  Fu- 
siform bacilli 
other  bacilli  accompanying,  613 
spirillum  variety  of,  613 
symptoms  of,  610 
Vincent's  spirilla,  staining  of,  108 
Virulence,  definition  of,  183 

determination   of,   in   diphtheria  ba- 
cilli, 519 
problem  of,  291 

variations  in,  and  infectiousness,  183 
Virulent  bacteria,  sublethal  doses  of,  in 

immunization,  195 
Virus  fixe,  in  specific  therapy  of  rabies, 
652 


Wadsworth's  method  of  staining  cap- 
sules, 99 
Wassermann  test  for  diagnosis  of  syph- 
ilis, 263 
antigen  for,  263 

determination  •  of   necessary   quan- 
tity of,  265 
obtaining    of,    from    alcoholic    ex- 
tracts of  syphilitic  organs,  263 
from  alcoholic  solution  of  normal 

organs,  263 
from  salt  solution  of  syphilitic 
liver,  263 
of  syphilitic  spleen,  263 
preparation  of,  by  Noguchi  meth- 
od, 266 
complement  in,  267 
hemolytic  serum  in,  265 
obtaining  of,  265 
potency  of,  265 
quantity  of,  265 
unit  in,  definition  of,  265 
determination  of,  265 
modifications  of,  268 


INDEX  OF  SUBJECTS 


851 


Wassermann  test  for  diagnosis  of  syph- 
ilis,   modifications    of,    Bauer's, 
269 
Noguchi's,  269 
performed    with    Spirochaeta    pallida 

antigen,  604 
preparation  for,  263 
serum  to  be  tested  for  syphilitic  anti- 
body in,  267 
sheep  corpuscles  in,  267 
technique  of,  268 
Water,  bacteria  in,  689 
in  bacterial  cell,  21 
in  ground  waters,  691 
in  perennial  springs,  692 
in  wells,  691 
in  rain  and  snow,  690 
in  surface  waters,  690 
influence  of  rain  on,  690 
light  and  temperature  factors  in 

purification  of,  691 
self-purification  in,  690 
pathogenic,  689 
of  cholera,  689 
of  diarrheal  diseases,  689 
of  typhoid  fever,  689 
presumptive  colon  tests  for,  697 
qualitative  analysis  of,  694 

isolation  of  cholera  vibrio  in,  695 

Koch's  method  of,  696 
isolation  of  colon  bacillus  in,  696 
isolation  of  typhoid  bacillus  in, 
694 
Adami's  and  Chapin's  method 

of,  695 
Drigalski's  method  of,  695 
Vallet's  method  of,  695 
quantitative  estimations  of,  692 
collecting  of  specimens  for,  692 
colon  bacilli  in,  696 
colon  test  in,  697 

counting  of  bacilli  in,  698 
counting  in,  694 
incubation  of  specimens  in,  694 
plating  of  specimens  in,  692 
value  of,  694 
bacteriological  examination  of, 
693 


Weil's  disease,  617 

Welch,  discovery  of  Bacillus  aerogenes 

capsulatus  by,  471 
Welch's  method  of  staining  capsules,  98 
Welch's     modification     of     Guarnieri's 

medium,  129 
Wertheim's  medium  for  cultivation  of 

gonococcus,  381,  382 
Winckel's  disease  in  the   newborn  due 

to  colon  bacillus,  394 
Wires  used  in  transferring  bacteria,  141 
Wolffhugel  counting  plate,  162 
Wool-sorter's  disease,  573 
Wright,  method  of,  of  anaerobic  culti- 
vation of  bacteria,  150 

modification  by,  of  Buchner's  pyro- 
gallic  method  of  cultivation  of 
anaerobic  bacteria,  153 

theory  of  opsonins  of,  282 

vaccine     therapy     of.     See    Vaccine 
therapy  of  Wright 
Wright's  stain,  796 


Xerosis  bacillus,  524 


Yaws,  614 

Yeast  cells,  cultivation  of,  633 
demonstration  of,  632 
morphology  of,  629 
reproduction  in,  by  budding,  629 
by  spore  formation,  630 
Yeasts,   629 

differentiation  of,  from  other  microor- 
ganisms, 629 
fermentation  by,  630 
industrial    employment    of,    for    fer- 
mentative purposes,  52 
infection  of,  in  animals,  682 
in  man,  631 

clinical  picture  of,  632 
pathogenic  varieties  of,  633 
Yellow  fever,  668 

clinical  picture  of,  668 
distribution  of,  668 
etiology  of,  668 
immunity  of,  674 


852 


INDEX  OF  SUBJECTS 


Yellow  fever,  investigation  of,  by  Gui- 
teras    and    Marchoux,    Salim- 
beni  and  Simond,  673 
results  of,  673 
by  Reed,  Carroll,  Agramonte,  and 
Lazear,  670 
results  of,  673 
microorganism  of,  biological  proper- 
ties of,  669 
research  for,  668 

by  Cornil  and  Babes,  669 
by  Sanarelli,  669 
by  Sternberg,  669 
Stegomyia  fasciata  in,  673 
description  of,  673 
power    of    transmission    of    infec- 
tion by,  reasons  for,  674 


Yellow    fever,    Stegomyia    fasciata    in, 
tropical     countries     most     fa- 
vorable for,  674 
transmission  of,  670 
by  mosquitoes,  670 

discovery  of,  by  Finlay,  670 
investigation     and     confirmation 
of,  by  United  States  Commis- 
sion, 670 
Yersin,  discovery  of  plague  bacillus  by, 
555 

ZuR  Nedden's  bacillus,  547 

cultivation  of,  547 

morphology  and  staining  of,  547 

pathogenicity  of,  547 
Zymase,  51,  630 


(11) 


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